Methods for producing caprylic acid and/or caprylate

ABSTRACT

Methods and systems to produce product compositions comprising caprylate products using chain-elongating bacteria. For example, the caprylate product in the product composition is n-caprylic acid (C8) and the n-caprylic (C8) to n-caproic (C6) acid ratio is higher than 1:1. These methods use chain elongation towards C8 rather than C6. High n-caprylate productivity and specificity was accomplished by: 1) feeding a substrate with, for example, ethanol as the carbon source or alternatively, a high ethanol-to-acetate ratio as the carbon source; 2) extracting caprylate product(s) (e.g., n-caprylate product) from the bioreactor broth; and 3) acclimating an efficient chain-elongating microbiome. The methods can produce caprylate products such as, for example, n-caprylic acid, which is a higher value chemical than C4 and C6.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/341,910, filed on May 26, 2016, the disclosure of which is herebyincorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under contract no.W911NF-12-1-0502 awarded by the U.S. Army Research Office and award#1336186 awarded by the National Science Foundation (SusChEM Program).The government has certain rights in the invention.

FIELD OF THE DISCLOSURE

The disclosure generally relates to biological production of caprylicacid or caprylate. More particularly the disclosure generally relates toproduction of caprylic acid or caprylate using a microbiome.

BACKGROUND OF THE DISCLOSURE

Ethanol as a biorefinery end product has shortcomings, including its lowvalue and high-energy needs for extraction. Concomitantly, researchersand engineers have developed a chain-elongation platform with opencultures (reactor microbiomes) with the goal of producing medium-chaincarboxylates (MCCs), such as n-caproate (C6) and n-caprylate (C8), fromethanol and short-chain carboxylates. MCCs have a higher value and areeasier to extract than ethanol due to their hydrophobic characteristicswhen in their undissociated chemical form. The longer the chain, themore value the product will have.

Anaerobic reactor microbiomes are capable of converting organic wastesto medium chain carboxylates via a process termed reversebeta-oxidation. Generally, an electron-rich substrate, such as onecontaining ethanol or lactic acid, is used to drive the chain elongationof shorter chain carboxylates, such as acetate and n-butyrate, to longerchain carboxylates, such as n-caproate or n-caprylate.

Medium-chain carboxylates (MCCs, ranging from six to 12 carbons), suchas n-caproate (C6), n-heptanoate (C7), and n-caprylate (C8), can beproduced within the carboxylate platform by chain elongating SCCs, suchas acetate (C2), propionate (C3), and n-butyrate (C4), via the reverseβ-oxidation pathway by adding two carbons during each cycle. There are avariety of different processes which result in medium chain and shortchain carboxylates, but the use of bioreactors and reverse betaoxidation seems particularly promising as a sustainable platform.Reverse beta oxidation is a metabolic pathway found in prokaryotes wherecarboxylate chains are reduced by the addition of two carbons from asubstrate, the best studied of which is probably ethanol. Since chainsare elongated two carbons at a time, an acetate molecule could go tobutyrate, which could go to caproate, which could go to caprylate,however this progression to caprylate does not occur or results inrather low levels. Common substrates that have been demonstrated inusing reverse beta oxidation include lactate, carbohydrates, andethanol.

Clostridium kluyveri, which is the type strain for the reverseβ-oxidation pathway, has been well studied to ascertain the mechanisticunderstanding of this pathway. In several studies with microbiomes themain product has been the even-chain MCC n-caproate, while small amountsof n-caprylate have been co-produced. Reactor microbiomes have alsoproduced the uneven-chain MCC n-heptanoate, albeit at a lowerselectivity (product vs. consumed substrate) than for n-caproateproduction. MCCs can be utilized as antimicrobial agents in agriculture;intermediates for fragrances and flavors; and precursors for renewablediesel fuel and aviation fuel. In all of these markets, a premium isavailable for longer-chain products (e.g. n-caprylic acid vs. n-caproicacid) due to their increased hydrophobicity and energy density.

Until now, the literature has described chain elongation processes towork by elongating dilute ethanol and acetate into n-caproate(6-carbons; C6), but with n-caprylate as a minor component side product,if produced at all. The value of n-caprylate is about twice that ofcaproate on a weight basis. Since n-caprylate is considerable morevaluable than n-caproate by weight, the question was raised whethermainly n-caprylate could be produced.

SUMMARY OF THE DISCLOSURE

In an aspect, the present invention provides methods of producingcaprylate/caprylic acid. The methods are based on reaction of a carbonsubstrate (e.g., ethanol or an ethanol/acetate mixture) with amicrobiome that has been acclimated to produce caprylate/caprylic acid.The methods are also based on removal of a portion of or all of thecaprylate/caprylic acid during the acclimation phase and/or productionphase. In an example, a method for producing a product compositioncomprises caprylate(s) (e.g., n-caprylic acid, n-caprylate, or acombination thereof) comprising an acclimation phase, a productionphase, and, optionally, one or more selection periods.

In an example, a method for producing a product composition comprisingcaprylate(s) comprises an acclimation phase, a production phase, and,optionally, one or more selection periods. For example, a methodcomprises: providing a reaction medium comprising one or morechain-elongating bacteria species, which may be present as all of or aportion of a microbiome having a pH of 5-8; adding substrate comprisingethanol or a mixture of ethanol and acetate; holding the reaction mediumat a desired temperature for a desired times during an acclimation phaseuntil the reaction mixture produces a desired about of n-caprylic acidor n-caprylate; continuously removing at least a portion or all of thecaprylic acid formed in the reaction medium during the acclimationphase, where the reaction medium is maintained at a pH of 5-8 during theholding and, optionally, continuously removing, where after theacclimation phase the reaction mixture produces a compositioncomprising, for example, at least 0.01% by weight caprylic acid in thereaction medium based on the total weight of the reaction medium, andcontinuously removing during a production phase at least a portion orall of the n-caprylic acid or n-caprylate formed in the reaction mediumto form the product composition.

In another example, a method comprises: providing a reaction mediumcomprising an acclimated microbiome or one or more chain-elongationbacteria; holding the reaction medium at a desired temperature andmaintaining the reaction medium a pH of 5-8, and continuously removingduring a production phase at least a portion or all of the n-caprylicacid or n-caprylate formed in the reaction medium to form the productcomposition. The pH of the reaction medium can be held at 5-8 during theproduction phase.

Additional substrate can be added to the reaction mixture (e.g., duringthe acclimation phase and production phase, and, if carried out, duringone or all of the selection periods). In various examples, additionalsubstrate is added periodically added, continuously added, or acombination thereof during one of more of the periods.

A method can comprise one or more selection periods. During theselection period the amount of caprylate in the reaction mixture isallowed to increase (e.g., built up) such that microbiome constituentsthat cannot tolerate the increased amount of caprylate do not surviveand microbiome constituents that tolerate the increased amount ofcaprylate (do not die) are increased in the microbiome. The resultingmicrobiome exhibits desirable production of caprylate. For example, aselection period comprises decreasing or stopping removal of caprylatefrom the reaction mixture (e.g., by decreasing or stopping extraction ofcaprylate from the reaction mixture).

A method can comprise a production phase. During a production phasecaprylate products are formed in the reaction medium. A productcomposition is formed by removal (e.g., by liquid extraction) of productcompounds from the reaction mixture. A product composition comprises oneor more caprylate.

In an aspect, the present invention provides systems for producingcaprylic acid. The systems comprise a continuous extraction system(e.g., an in-line continuous extraction system). Examples of systemsinclude, but are not limited to, anaerobic upflow bioreactors comprisinga continuous extraction system (e.g., an in-line continuous extractionsystem). The systems can carry out a method of the present disclosure.Examples of systems are provided herein.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

For a fuller understanding of the nature and objects of the disclosure,reference should be made to the following detailed description taken inconjunction with the accompanying figures.

FIG. 1 shows examples of carboxylate productivities and product ratiosof n-caprylate to n-caproate. Medium-chain carboxylate (MCC)productivities increased to 21.1 g COD/L-d by increasing organic loadingrates (OLRs) up to 34.7 g COD/L-d. A higher OLR resulted in a stagnatedMCC productivity. n-Caprylate (green) was the predominant product, witha product ratio of n-caprylate to n-caproate of 25 g COD/g COD (22.7 byweight) at an OLR of 15.0 g COD/L-d, and a product ratio of 11 g COD/gCOD (10 by weight) at an OLR of 34.7 g COD/L-d. Error bars represent 95%confidence intervals. Data shown are from Phase II.

FIG. 2 shows n-caprylate productivities from bioreactors with ethanol asan electron donor, including Phase II. Results from previously publishedstudies for which n-caprylate production was reported (small squares andX). Also shown are results from this Example (large squares). Operatingperiods from Phase II of the present Example are labeled with whitefont. Maximum instantaneous values reported are indicated (*). Thereferenced study (see main text), the organic loading rate (OLR), andthe n-caprylate productivity are listed. Both n-caprylate productivitiesand OLRs are presented on logarithmic scales. One study producedn-caprylate in a bioreactor in which gas composed of carbon dioxide andhydrogen was fed; they did not report OLRs, so this marker was placed atan OLR near the sum of the total carboxylate volumetric productionrates. Our study achieved maximum n-caprylate productivities up to 19.4g COD/L-d (Period 10).

FIG. 3 shows examples of bioreactor broth concentrations, organicloading rates, and medium-chain carboxylate productivities.Concentrations carboxylates (A) and of ethanol (B) in the bioreactorbroth were determined from samples collected every other day or daily.Average medium-chain carboxylate (MCC) productivities (A) and totalorganic loading rates (OLRs) (A) for each operating period are alsoshown. A color gradient was used to show the product ratio ofn-caprylate (green) to n-caproate (purple) with blue representing amixture of these two products. Operating phases, operating periods, andpresence (+) or absence (−) of product recovery via in-line pertractionare indicated. Detection limits were 0.05 g COD/L (0.5 mM) for ethanoland ˜0.02 g COD/L (˜0.1 mM) for carboxylates.

FIG. 4 shows product ratios and substrate ratios for a batch experimentwith microbiomes. Increased substrate ratios of ethanol to acetate ledto increased product ratios of n-caprylate to n-caproate. The totalaccumulated MCC concentration is indicated by the size of the circle andthe corresponding black text of n-caproate plus n-caprylate in g COD/L(pH 5.4±0.1, 12-d incubation). Four initial concentrations of ethanolwere evaluated. In general, a higher initial concentration of ethanolled to a lower product ratio. Substrate inhibition was apparent at 28.8g COD/L (300 mM) ethanol for all substrate ratios.

FIG. 5 shows a heat map of relative abundances for 36 OTUs in 11 reactormicrobiome samples during the high n-caprylate productivity of Phase II.During Phase II, each of the 36 operational taxonomic units (OTUs)listed comprised at least one percent of the relative abundance for oneor more of the microbiome samples collected. OTUs were clusteredhierarchically (average linkage) based on the Bray-Curtis dissimilarityindex. OTUs were grouped together based on both the average relativeabundance and abundance profile. This resulted in the localization ofOTUs with lower abundances in the top half, and OTUs with higherabundances in the bottom half of the heat map. An Acinetobacter spp.increased in dominance up to 55.5% of the relative abundance.Subsequently, a Rhodocyclaceae K82 spp. became dominant, comprising upto 70.8% of the relative abundance. Relative abundances of five OTUs(asterisks) were correlated (p<0.05) with n-caprylate productivities.

FIG. 6 shows beta diversity and constrained ordination for 11 microbiomesamples during the high n-caprylate productivity of Phase II. Principalcoordinates analysis (PCoA) (A) and capscale analysis (dbRDA) (B) wereperformed with sequence and performance data for 11 reactor microbiomesamples. The increasing blue color of the circles for the 11 samplesindicates the increasing length for the operating period of Phase II,including Day 106, 120, 126, 134, 140, 150, 155, 163, 174, 176, and 186.The five lighter blue circles (lower left quadrants) represent samplesfrom early in Phase II (Periods 6-7), while the four darkest circles(right half) represent samples from the end of Phase II (Period 10-11).Average residual ethanol concentrations in the bioreactor broth andaverage hydraulic retention times (HRTs) were not collinear (VIF<5), butthey were significant variables (p<0.05) that explained 88% of thevariation seen in the PCoA.

FIG. 7 shows the reverse β-oxidation pathway. With the addition ofethanol, short-chain carboxylates (e.g. acetate) can be chain elongatedto medium-chain carboxylates (e.g. n-caprylate).

FIG. 8 shows a schematic of an example of a bioreactor system. An upflowanaerobic filter was continuously fed with ethanol and acetate. In-lineproduct extraction was used to continuously recover hydrophobic,undissociated MCCAs from a bioreactor broth recycle flow through theforward membrane contactor. After intermediary recovery in a mineral oilsolvent, MCCAs were then transferred across a second, backward membranecontactor to an alkaline extraction solution. Through automatic baseaddition to the alkaline extraction solution, the pH gradient wasmaintained, and these products accumulated in the alkaline extractionsolution as MCCs.

FIG. 9 shows biogas composition during Phase II. Hydrogen was quantifiedusing the reduced gas detector at concentrations beneath 2000 ppm, whileit was quantified with a gas chromatograph at higher concentrations.Methane concentrations were undetectable until Period 11 in Phase II,and the methane increase corresponded with increasing hydrogenconcentrations and undetectable carbon dioxide concentrations.

FIG. 10 shows substrate ratios and concentrations affected carboxylateproduct ratios and concentrations in batch and continuously fedbioreactors of C. kluyveri. For (A)-(C), the concentrations of ethanoland carboxylates that were either produced (positive values) or consumed(negative values) are shown for batch bioreactor experiments of C.kluyveri. For (D), the net volumetric production rate (productivity) ofethanol and carboxylates that were either produced (positive values) orconsumed (negative values) are shown for a continuously fed bioreactorof C. kluyveri. In all experiments, ethanol and acetate were fed. Theinitial substrate ratio (ethanol to acetate) for each treatment isdisplayed upon the concentration of the ethanol consumed. In addition,the product ratio (n-caproate to n-butyrate) for each treatment isdisplayed upon the concentration of the n-caproate produced. Morespecifically: (A) in this batch study, the initial concentration ofethanol was fixed (4.5 g COD/L, 47 mM) and the initial concentration ofacetate was varied. The bioreactor temperature was 30° C., the pH was 7,and the duration was 12 d. When the initial concentration of acetate wasincreased, the ratio of n-caproate to n-butyrate produced decreased. Atthe maximum initial concentration of acetate fed, the producedn-caproate concentration decreased; (B) in this batch study, the initialconcentration of ethanol was fixed (33.6 g COD/L, 350 mM) and theinitial concentration of acetate was varied. The bioreactor temperaturewas 39° C., the initial pH was 6.8, and the duration was 3 d. When theinitial concentration of acetate was increased, the ratio of n-caproateto n-butyrate produced decreased. At the maximum initial concentrationof acetate fed, the produced n-caproate concentration decreased; (C) inthis batch study, the initial concentration of acetate was fixed (7.7 gCOD/L, 120 mM) and the initial concentration of ethanol was varied. Thebioreactor temperature was 39° C., the initial pH was 6.8, and theduration was 3 d. When the initial concentration of ethanol wasincreased, the ratio of n-caproate to n-butyrate produced increaseduntil the initial concentration of ethanol was 44 g COD/L (460 mM);higher initial levels of ethanol led to substrate inhibition anddecreased ethanol utilization; and (D) in this continuously fedbioreactor study, the substrate ratio of ethanol to acetate was eitherethanol-limited or had excess ethanol (2 or ˜7 g COD/g COD,respectively, which is 1.04 or ˜3.63, respectively, by weight [dividedby 1.927]). Increased substrate ratios and decreased substrateconcentrations led to increased product ratios.

FIG. 11 shows substrate ratios and ethanol concentrations affected MCCproduct ratios and concentrations in batch reactor microbiomes. Theconcentrations of carboxylates that were either produced (positivevalues) and the ethanol and acetate that were consumed (negative values)are shown for three batch experiments with reactor microbiomes that weperformed. In all experiments, ethanol and acetate were fed, and eachconcentration represents the average of triplicate biological batchbottles. The temperature of the bioreactors was controlled at 30° C. andthe initial pH was set at approximately 5.4 with an experimental periodof 12 d. The initial substrate ratio (ethanol to acetate) for eachtreatment is displayed in white font within the pink bar for the ethanolconcentration consumed, while the product ratio (n-caprylate ton-caproate) for each treatment is displayed in white font in the centerof the green bar for the n-caprylate concentration produced. Morespecifically: (A) the initial concentration of ethanol was fixed (9.6 gCOD/L, 100 mM) and the initial concentration of acetate was varied. Whenthe initial concentration of acetate was increased (which consequentlydecreased the initial substrate ratio of ethanol to acetate), theproduct ratio of n-caprylate to n-caproate decreased. Increasedsubstrate ratios of ethanol to acetate led to increased n-caprylateproduct ratios; (B) the initial substrate ratio of ethanol to acetatewas fixed (13.5 g COD/g COD [7.0 by weight]) and the substrate levelswere varied. At this fixed substrate ratio, the lower substrateconcentrations resulted in the higher product ratios of n-caprylate ton-caproate, as well as the higher concentrations of n-caprylate. Atinitial ethanol concentrations of 28.8 g COD/L (300 mM), considerablesubstrate inhibition of medium-chain carboxylate production wasobserved; and (C) the initial acetate concentration was fixed (˜0.7 gCOD/L, ˜10 mM) and the initial concentrations of ethanol were varied. Aninitial concentration of ethanol of 28.8 g COD/L (300 mM) led tosubstrate inhibition of chain elongation, even with fixed acetateconcentrations.

FIG. 12 shows an example where the overall mass transfer coefficient wasdirectly proportional to the abiotic reactor broth recycle flow rate.During an abiotic n-caproate transfer experiment that used a similarpertraction system (same materials, but a different size of contactors)than this bioreactor experiment, we determined that the overall masstransfer coefficient (k) was directly proportional to the reactor brothrecycle flow rate (r). Each of the contactors can be compared whencorrected for the superficial velocity (u). Increasing the recycle flowrates of mineral oil solvent or the alkaline extraction solution did notaffect mass transfer rates, indicating that mass transfer limitationswere at the interface of the reactor broth and the hydrophobic membranecontactor. The overall mass transfer coefficient was linearly correlatedto the reactor broth recycle flow rate through the highest flow ratesthat the pumps could provide (690 L/d). During the continuously fedbioreactor experiment, however, we maintained a constant recycle flowrate (r), mass transfer coefficient (k), and membrane surface area (A).With these fixed values fixed, improvements in MCC transfer andproduction rates could only be achieved by higher concentrations ofundissociated medium-chain carboxylic acids (MCCAs) in the bioreactorbroth. The data here shows that we could have increased the MCC transferrates with the same membrane contactors if we had increased thebioreactor broth recycle flow (but we did not increase it).

FIG. 13 shows undissociated n-caprylic acid concentrations from examplesof bioreactors with ethanol as an electron donor, including Phase II.Results from previously published studies in which n-caprylateproduction was reported are shown. Also shown are results from thisExample (large squares). Operating periods from Phase II of this Exampleare labeled with a white font. Maximum instantaneous values reported areindicated (*). Organic loading rates are presented on logarithmicscales. One study produced n-caprylate in a bioreactor in which gascomposed of carbon dioxide and hydrogen was fed; they did not reportOLRs, so this marker was placed at an OLR near the sum of the totalcarboxylate volumetric production rates. The highest concentration ofundissociated n-caprylic acid concentrations during Period 11 of ourExample likely led to product inhibition.

FIG. 14 shows 48 OTUs with a relative abundance that exceeded 1% of atleast one microbiome sample during the entire operating period. Relativeabundances of operational taxonomic units (OTUs) varied during theoperating period. Dominant OTUs included Rhodocyclaceae K82 spp. andAcinetobacter spp., which comprised up to 70.8 and 55.5% of the relativeabundance, respectively. Phylogenetic similarity is indicated.

FIG. 15 shows alpha diversity of reactor microbiome sample during theoperating period. The Shannon index was used to determine the evennessand richness for the 16 reactor microbiome samples that we collected,including the inoculum. Uncertainty is represented by 95% confidenceintervals based on ten independent rarefactions.

FIG. 16 shows beta diversity of reactor microbiome samples during anentire operating period. Principal coordinates analysis (PCoA) was usedto determine the dissimilarity between microbiome samples taken based onthe weighted UniFrac metric. The first two principal coordinate (PC)axes are shown. PC1 explains 43% of the overall phylogenetic variation,while PC2 explains 24%. The increasing blue color of the circles for the16 bioreactor samples indicates the increasing length for the operatingperiod when the sample was taken, including Day 17, 30, 52, 78, 94, 106,120, 126, 134, 140, 150, 155, 163, 174, 176, and 186. The white squarerepresents the inoculum.

FIG. 17 shows accumulation of medium chain carboxylates (MCCs) in thestripping solution overtime at the fixed ethanol to acetate molar feedratio of 10:1 (12.8:1 by weight). C8 is caprylate and C6 is caproate.This ratio gave the highest transfer and overall production rates ofMCCs for the entire Example.

FIG. 18 shows the concentrations of various carboxylates (in g COD/L) ofthe reactor effluent over the course of the 10:1 molar feed ratio(12.8:1 by weight).

FIG. 19 compares the conversion efficiencies of the 10:1 molar ethanolto acetate feed ratio (12.8:1 by weight) in three experiments. Overallconversion of both caproate and caprylate is higher in the leftreplicant, however the ratio of caprylate to caproate is higher in thetwo right replicants. The hydraulic retention time (HRT) for each trialvaries. The HRT of the left replicant was 1.32±0.10 days (a 95%confidence interval), whereas the other HRTs were 1.5 days and 3.3 daysrespectively. Error bars are not displayed in this figure, though can befound in FIG. 25. Order of data within the bars: Top (C8), bottom (C6)

FIG. 20 shows effluent concentrations of different carboxylates overtime from a 10:0, pure ethanol, feed.

FIG. 21 shows total carboxylates found in the stripping solution overtime for the different the ethanol to acetate ratios tested in the feed.Each color change represents the change in feed ratios in descendingorder, starting from 10:0 and going to the beginning of the 6:4 run. Ascan be seen clearly in figure, there are quite noticeable changes inslope (a function of the transfer rate) over time. Initially, at the tento zero ethanol to acetate ratio, a majority of the carboxylatesaccumulating in the feed are caprylate (indicated by C8). As the ratiosgo down, so too does the transfer rate of caprylate. From the 8:2 ratioonwards, butyrate (But) started to be detected by the GC, and as theratios decrease the transfer rate of butyrate goes up. The caproate (C6)slope initially increases and then decreases as the ratio of ethanol toacetate decreases further. By 6:4, acetate started to be found in thestripping solution in low concentrations (not plotted). At the time ofthis paper, only 4 data points for 6:4 have been run in the GC.

FIG. 22 shows concentration of carboxylates found in examples ofeffluent in gCOD/L. Each color change corresponds to a change in theinfluent molar ratio of ethanol to acetate. To find the effluent rates,the averages of each carboxylate were taken over the course for eachrespective feed ratio and divided by the hydraulic retention times ofeach phase. General trends in this figure show that as the ethanol toacetate feed ratio decreases, the concentration of the MCCs in thereactor goes down as the concentration of short chain carboxylates(butyrate and acetate) goes up.

FIG. 23 shows using the same data as that found in FIG. 23, howevercaprylate is isolated and the y-axis is rescaled to clearly show thecaprylate ratios in gCOD/L in the reactor broth.

FIG. 24 shows using the same data as that found in FIG. 23, howevercaproate is isolated and the y-axis is rescaled to clearly show thecaproate ratios in gCOD/L in the reactor broth. Of note is the decreaseof caproate during the pure ethanol run (leftmost of the figure).

FIG. 25 shows productions rates of only the MCCs for different molarfeed ratios. The error bars represent the 95% confidence intervals. Thenumbers in each of the bars are the ratios of caprylate production ratesto caproate production rates. The organic loading rate for each periodis approximately 25 gCOD/L-day. The overall production of MCCs washighest after the initial period at 8:2 ethanol to acetate, though theratio of caprylate to caproate continues to decrease throughout thetrials. Order of data within the bars: Top (C8), bottom (C6).

FIG. 26 shows specificities of each of the major carboxylates producedby the reactor. Absent is acetate and odd numbered carbon carboxylates,the latter of which was not found consistently in the stripping oreffluent. The trend of increasing butyrate, decreasing caprylate, andincreasing then decreasing caproate can be seen again. Order of datawithin the bars: Top (butyrate), middle (C8), bottom (C6).

FIG. 27 shows concentration of ethanol in the reactor broth over timeand influent molar ratios. It should be noted that this scale is inmolar terms as opposed to gCOD/L. It should also be noted that there isa considerable gap between the last data point of the pure ethanol trialand the 9:1 trial. This is due to missing data that should be filled inat a later date. The overall trend shows a dramatic increase in ethanolconcentration during the pure ethanol phase which then steadilydecreases in subsequent periods of lower ethanol to acetate feed ratios.

FIG. 28 shows conversion efficiencies of each of the major products ofthe bioreactor for the different trials. Conversion efficiency is theratio of the production rate of each carboxylate over the organicloading rate of the feed. The error bars in the figure represent 95%confidence intervals. At higher ethanol to acetate ratios, caprylateconversion is larger than caproate and butyrate conversion. As theinfluent ratio decreases, the total conversion increases, but caprylateconversion goes down. Butyrate conversion goes up, and caproateproduction first increases and then decreases. Order of data within thebars: Top (butyrate), middle (C8), bottom (C6).

FIG. 29 shows the pertraction efficiencies of carboxylates at varyingfeed ratios and over time. The pertraction efficiency is the fraction ofthe total production rate that is accounted for by the transfer rate. Inthis sense it is a measure of the efficiency of the stripping system.The pertraction efficiency for caprylate remains over 95% for all butthe last period. The high efficiency implies that the production rate isnot limited by the mass transfer of MCCs into the stripping, especiallyfor caprylate. The figure also shows that the pertraction efficiency ofcaproate and butyrate increases with lower ethanol to acetate feedratios. Order of bars left to right: Left (C6), middle (C8), right(butyrate). Butyrate not produced in 10:1, 10:0 and 9:1 (these are molarratios).

FIG. 30 shows varying gas compositions over the course of theexperiment. The gas production rate was very low, the data is notincluded in this figure. The detection limit of the Gas GC for carbondioxide, methane, and nitrogen was about 1%. The lower detection limitof hydrogen was about 0.2%. After the 9 to 1 ethanol to acetate ratio,hydrogen becomes undetectable. The numbers in the bars show the averagesof the composition. Order of data within the bars: Top* (hydrogen),next⁺(CO₂), third (methane), bottom (nitrogen). * The Hydrogen ‘bar’ isnot present in 8:2, 7:3, 6:4 and 5:5 (based on moles). ⁺ The CO2 ‘bar’is not present in 8:2 (based on moles).

FIG. 31 shows production rate (A) and effluent concentration (B) ofcarboxylates in 5 phases with different bioreactor pH (6.75, 6.25, 5.5,6.0 and 7.0).

FIG. 32 shows molar percentage of caproic acid and caprylic acid inMCCAs oil.

FIG. 33 shows generalized stoichiometric model for the fermentation ofethanol and acetate to n-butyrate, n-caproate, n-caprylate, andmolecular hydrogen by Clostridium kluyveri. This model is the extendedversion of a previously developed model. The variable “a” representsmoles of ethanol, “b” represents moles of n-butyrate, “c” representsmoles of n-caproate. Redox factors are highlighted in blue; classicalenergy conservation in red; and more recently described mechanisms ofenergy conservation in yellow. F₀/F₁ is H⁺/Na⁻-pumping ATP synthasecomplex and Rnf is the ferrodoxin-NAD reductase complex.

FIG. 34 shows COD specificity (gCOD carboxylate/g COD othercarboxylate(s)) of carboxylate produced at each substrate molar ratioduring the main periods of the Example (Periods 1 to 7) for n-butyrate,n-caproate, and n-caprylate.

FIG. 35 shows effluent concentrations of carboxylates (A) and ethanol(B) during the operating period in the bioreactor. Shaded sectionsrepresent even periods (i.e., Period 2, Period 4, Period 6).

FIG. 36 shows a heat map of relative OTU abundances in biomass samplestaken from the bottom port of the bioreactor during the main periods ofthe Example (Periods 1 to 7). Sampling day numbers are shaded grey torepresent even periods (Periods 2, 4, and 6). Relative abundancefraction is represented by the color gradient shown. OTUs that reachedover 1% relative abundance in any one sample are represented, resultingin 40 OTUs. OTUs are clustered hierarchically (average linkage) based onthe Bray-Curtis dissimilarity index. Lowest level taxonomy names as wellas OTU IDs are provided. Blue shading represents OTUs that are unique tobottom of the bioreactor (i.e., not found in the samples taken from themiddle of the bioreactor). + or − symbols represents whether therelative abundance of the OTU was found to be significantly positively(+) or negatively (−) correlated with n-caprylate specificities based onSpearman's rank correlation coefficient (p<0.001).

FIG. 37 shows a heat map of relative OTU abundances in biomass samplestaken from the middle of the bioreactor during the main periods of theExample (Periods 1 to 7). Relative abundance fraction is represented bythe color gradient shown. OTUs that reached over Sampling day numbersare shaded grey to represent even periods (Periods 2, 4, and 6). 1%relative abundance in any one sample are represented, resulting in 40OTUs. OTUs are clustered hierarchically (average linkage) based on theBray-Curtis dissimilarity index. Lowest level taxonomy names as well asOTU IDs are provided. Blue shading represents OTUs that are unique tomiddle of the bioreactor (i.e., not found in the samples taken from thebottom of the bioreactor). + or − symbols represent whether the relativeabundance of the OTU was found to be significantly positively (+) ornegatively (−) correlated with n-caprylate specificities based onSpearman's rank correlation coefficient (p<0.001).

FIG. 38 shows Gibbs free energy of an example of a reaction vs.ethanol-to-acetate molar ratio measured in bioreactor. Gibbs free energyof the reaction based on the ethanol and carboxylate concentrationsmeasured in the bioreactor is plotted as delta G produced. Delta Grequired is calculated based on the amount of ATP produced based on thestoichiometric model and assuming that −72 kJ is required per mole ofATP produced. Numbers on figure indicate the substrate molar ratios thatwere fed into the bioreactor, for the higher substrate molar ratios.

FIG. 39 shows the relative abundance of the phyla Firmicutes andProteobacteria in the samples collected from the bottom (A) and middle(B) sampling ports of the bioreactor.

DETAILED DESCRIPTION OF THE DISCLOSURE

Although claimed subject matter will be described in terms of certainembodiments, other embodiments, including embodiments that do notprovide all of the benefits and features set forth herein, are alsowithin the scope of this disclosure. Various structural, logical,process step, and electronic changes may be made without departing fromthe scope of the disclosure.

Ranges of values are disclosed herein. The ranges set out a lower limitvalue and an upper limit value. Unless otherwise stated, the rangesinclude all values to the magnitude of the smallest value (either lowerlimit value or upper limit value) and ranges between the values of thestated range.

The present disclosure provides methods for producing caprylic acid. Thepresent disclosure also provides systems for producing caprylic acid.

Our previous work produced mainly C6, and when we considered if we couldmainly produce C8 rather than C6, we did not consider it possible. Theresults presented herein were surprising, especially the greater amountsof C8 relative to C6 produced.

Caprylate is a carboxylate, and herein this term is used to include boththe dissociated species (caprylate) and the undissociated species(caprylic acid). Caprylic acid and caprylate are also known asn-octanoic acid and n-octanoate. Caprylate can have various countercations. Examples of suitable cations include, but are not limited to,sodium ion, potassium ion, and the like. The present methods producemainly caprylate, the other carboxylates are at low productivities. Thistechnology uses microbiomes at, for example, ambient pressures, reducingcapital costs by ensuring a simple bioreactor design.

An anaerobic upflow bioreactor was used to produce levels of n-caprylateat levels that have not been previously reported. For one example, thisincrease in the n-caprylate productivity to 19.4 g chemical oxygendemand (COD)/L-d with a product ratio of n-caprylate to n-caproate of 11g COD/g COD. In another example, this ratio was 25 g COD/g COD at anearlier operating period though with a little lower productivity,resulting in a specificity of 96% when compared to all carboxylates. Weaccomplished this high n-caprylate productivity and specificity by: 1)feeding a substrate with ethanol as the sole carbon source oralternatively, a high ethanol-to-acetate ratio as the sole carbonsource; 2) extracting the n-caprylate product from the bioreactor broth;and 3) acclimating an efficient chain-elongating microbiome. Becausesyngas fermentation effluent consists of a high ratio of ethanol toacetate, these syngas fermentation product of ethanol and alternativelyhigh ratio of ethanol to acetate resulted in chain elongation to producen-caprylate.

Biomass is not a component of the instant methods/systems, unlike otherbioreactor and incubation systems. For example, ethanol is used as thecarbon source or, alternatively, ethanol and acetate is used as thecarbon substrate, where the ratio of ethanol:acetate is kept at a highlevel (e.g., at least at a 5:1 molar ratio).

We obtained the breakthrough of producing mainly caprylate due to acombination of operating conditions: 1) using ethanol as the solesubstrate or in some instance using high substrate ratio of ethanol toacetate; 2) the presence of in-line product extraction that is selectivefor longer-chain carboxylates; and 3) the adaptation of an efficientmicrobiome. Our bioprocess is versatile and can be coupled to existingfermenters to displace ethanol distillation.

In an aspect, the present invention provides methods of producingcaprylate/caprylic acid. The methods are based on reaction of a carbonsubstrate (e.g., ethanol or an ethanol/acetate mixture) with amicrobiome that has been acclimated to produce caprylate/caprylic acid.The methods are also based on removal of a portion of or all of thecaprylate/caprylic acid (referred to herein as extraction, pertraction,or stripping) during the acclimation phase and/or production phase. Inan example, a method does not comprise passing an electric currentthrough the reaction medium and/or electrical stimulation of themicrobiome or one or more chain-elongation bacteria.

In an example, a method for producing a product composition comprisingcaprylate(s) (e.g., n-caprylic acid, n-caprylate, or a combinationthereof) comprises an acclimation phase (e.g., Phase I describedherein), a production phase (e.g., Phase II described herein), and,optionally, one or more selection periods (e.g., period 5 in FIG. 3).For example, a method comprises: providing a reaction medium comprisingone or more chain-elongating bacteria species, which may be present asall of or a portion of a microbiome, having a pH of 5-8 (e.g., 5-7.5,5-7, 5-6, 5-5.55, 5.1-5.2, or 5.5); adding substrate comprising ethanolor a mixture of ethanol and acetate (e.g., an ethanol and acetatemixture having an ethanol:acetate molar ratio of 5:1 or greater);holding (e.g., at a temperature of 25 to 38° C.) the reaction mediumduring an acclimation phase (e.g., for at least 1 day) until thereaction mixture produces a desired about of n-caprylic acid orn-caprylate (an efficient chain elongation microbiome is acclimated);continuously removing at least a portion or all of the caprylic acidformed in the reaction medium during the acclimation phase, where thereaction medium is maintained at a pH of 5-8 (e.g., 5-7.5, 5-7, 5-6,5-5.55, 5.1-5.2, or 5.5) during the holding and, optionally,continuously removing, where after the acclimation phase the reactionmixture produces a composition comprising, for example, at least 0.01%by weight, or 0.05% by weight or 0.1% by weight caprylate(s) (e.g.,caprylic acid) in the reaction medium based on the total weight of thereaction medium, and continuously removing during a production phase atleast a portion or all of the n-caprylic acid or n-caprylate formed inthe reaction medium to form the product composition.

A method can used an acclimated microbiome or one or morechain-elongating bacteria species. For example, a method comprises:providing a reaction medium comprising an acclimated microbiome or oneor more chain-elongation bacteria; holding the reaction medium at adesired temperature (e.g., at a temperature of 25 to 38° C.) andmaintaining the reaction medium a pH of 5-8 (e.g., 5-7.5, 5-7, 5-6,5-5.55, 5.1-5.2, or 5.5), and continuously removing during a productionphase at least a portion or all of the n-caprylic acid or n-caprylateformed in the reaction medium to form the product composition.

A reaction mixture (which is also referred to herein as a broth)comprises one or more species of chain-elongating bacteria. Thechain-elongating bacteria can be present in the form of a microbiome.Various microbiomes or purified microbiomes can be used. Microbiomes canbe naturally occurring or engineered. For example, suitable microbiomesare obtained from anaerobic environments such as, for example, anaerobicdigester sludge, soil, aqueous sediments, the gut of animals, and thelike. A microbiome can include a diverse microbial community. It can bean open culture in which bacteria can enter with the substrate and forwhich sterilization is not needed. A microbiome comprises one or morechain-elongating bacteria species. Without intending to be bound by anyparticular theory, it is considered that a chain-elongating bacteriumforms a product compound (e.g., a caprylate product) by a reverseβ-oxidation pathway by adding two carbons during each cycle. Themicrobiome can comprise an even community or an uneven community. In anexample, a microbiome comprises 1-50 different chain-elongating bacteriaspecies, including all integer numbers of chain-elongating bacteriaspecies and ranges therebetween. Bacteria in the microbiome can beidentified by gene sequencing. For example, the most abundant bacteria(48 operating taxonomic units, which is a quantifiable number todescribe particular species of bacteria) in a microbiome are identifiedafter 16S rRNA gene sequencing (FIG. 5 and FIG. 14 in the Example 1). Inan example, a microbiome comprises 48 operating taxonomic units(bacteria species) described in FIG. 5 or FIG. 14 in Example 1.Microbiomes can produce various liquid and/or gaseous product compoundssuch as, for example, carboxylic acids, alcohols, aldehydes, hydrogen,carbon dioxide, methane, and the like. Examples of chain-elongatingbacteria and microbiomes are provided herein. Examples ofchain-elongating bacteria and microbiomes are known in the art. Examplesof chain-elongating bacteria and microbiomes are commercially available.

A microbiome can comprise additional components. For example, amicrobiome further comprises one or more components that facilitategrowth and/or stability of a microbiome. Suitable components are knownin the art. In an example, for growth, a microbiome comprises one ormore trace elements including, for example, metals, nutrients, vitamins,or a combination thereof.

It is desirable to control the pH of the reaction mixture. In variousexamples, the pH of the reaction mixture is maintained (e.g., during theacclimation phase and production phase, and, if carried out, during oneor all of the selection periods) at a pH of 3.0 to 8.0 (e.g., 5-7.5,5-7, 5-6, 5-5.55, 5.1-5.2, or 5.5), including all 0.1 pH units andranges therebetween.

It can be desirable to control the temperature of the reaction mixture.In various examples, the temperature of the reaction mixture ismaintained (e.g., during the acclimation phase and production phase,and, if carried out, during one or all of the selection periods) at 15°C. to 45° C. (e.g., 25 to 38° C. or 30° C.), including all 0.1 C valuesand ranges therebetween.

The reaction mixture can be present in various environments. Forexample, the reaction mixture is present in an inert environment (e.g.,under an inert gas such as, for example, nitrogen). The reaction mixturecan be present in an anaerobic environment. The reaction can be rununder ambient pressure (no pressurization is required) or under apressurized environment. In an example, the reaction mixture is under apressure of 0.7-1.3 atmospheres.

A reaction mixture comprises substrate. The substrate serves as a carbonsource. Examples of substrate include ethanol and ethanol/acetatemixtures. Various amounts of substrates can be used. In the case wherethe substrate is an ethanol/acetate mixture, it may be desirable thatthe mixture has an ethanol to acetate molar ratio of greater than 1 to 1or greater, 2 to 1 or greater, 4.5 to 1 or greater, 5 to 1 or greater,5.5 to 1 or greater, 6 to 1 or greater, 10 to 1 or greater, 25 to 1 orgreater, 50 to 1 or greater, or 100 to 1 or greater. In the case wherethe substrate is an ethanol/acetate mixture, the mixture has an ethanolto acetate molar ratio of 1:1 to 100:1, 4.5:1 to 25:1, 4.5:1 to 50:1, or4.5 to 100:1.

Additional substrate can be added to the reaction mixture (e.g., duringthe acclimation phase and production phase, and, if carried out, duringone or all of the selection periods). In various examples, additionalsubstrate is added periodically added, continuously added, or acombination thereof during one of more of the phases and/or periods. Itis desirable not to overfeed (feed more than the rate of chainelongation, which is equal to the extraction rate) the microbiome duringone or more or all of the phases and/or periods.

A method comprises an acclimation phase (also referred to herein as aperiod) during which certain bacteria outgrow others to achieve anacclimated microbiome. During the acclimation phase an efficient chainelongation microbiome is produced. By “efficient chain elongationmicrobiome” it is meant that a microbiome produces the longest possiblemedium-chain carboxylate that is possible under the environmentalconditions at desirable production rates. During the acclimation phase,the amount of one or more chain-elongating bacteria species areincreased relative to one or more other bacteria in the reactionmixture. During the acclimation phase, a portion of or all of thecaprylate product is removed (e.g., by in-line, continuous extraction asdescribed herein) from the reaction mixture. It is desirable to maintainthe caprylate product concentration at a level that is not toxic to oneor more constituents of the microbiome. Too high concentrations ofundissociated carboxylic acids (caprylate product(s)) can inhibitproduction of caprylate product(s). Accordingly, it is desirable tomaintain the amount of undissociated caprylate(s) in the reactionmixture at a level that does not inhibit production of caprylateproduct(s).

In an example, after an acclimation phase a reaction mixture (e.g.,microbiome) comprises one or more species of bacteria selected fromRuminococcus spp. (1818318)+, Rhodocyclaceae K82 spp. (1140386),Oscillospira spp. (115035), Acetobacter spp. (635373), and unknownBacteroidaes, and combinations thereof. In an example, after anacclimation phase a reaction mixture (e.g., microbiome) comprises (e.g.,predominantly comprises) one or more species of bacteria from theFirmicutes and/or Proteobacteria phyla. The one or more species ofbacteria may present at the same or different amounts in the reactionmixture.

During the acclimation phase, the reaction mixture is held at a desiredtemperature (e.g., 15° C. to 45° C., 25 to 38° C., or 30° C.) until thereaction mixture provides a desired level of caprylate product. Invarious examples, the reaction mixture is held at a desired temperature(e.g., 15° C. to 45° C., 25 to 38° C., or 30° C.) (e.g., at atemperature of 25 to 38 (30° C.)) for at least 1 day, at least 10 days,at least 25 days, at least 50 days, at least 100 days, at least 200days, or at least 400 days.

After an acclimation phase, the reaction mixture produces a desirableamount of (e.g., predominantly) caprylate product(s). In an example,after the acclimation phase, the reaction mixture produces caprylateproduct(s) so that the concentration of at least 0.01% by weight, atleast 0.05% by weight, or 0.1% by weight, or 0.5% by weight caprylateproduct(s) in the reaction medium based on the total weight of thereaction medium. In another example, after the acclimation phase, thereaction mixture produces caprylate product(s) so that the concentrationof at least 0.1 g/L or g COD/L, at least 0.5 g/L or g COD/L, or 1 g/L org COD/L caprylate product(s) in the reaction medium based on the totalweight of the reaction medium.

A method can comprise one or more selection periods. During a selectionperiod the amount of caprylate in the reaction mixture is allowed toincrease (e.g., built up) such that microbiome constituents that cannottolerate the increased amount of caprylate do not survive and microbiomeconstituents that tolerate the increased amount of caprylate (do notdie) are increased in the microbiome. The resulting microbiome exhibitsdesirable production of caprylate. Typically, the microbiome after aselection period exhibits increased production of caprylate(s) relativeto that exhibited by the microbiome prior to the selection period. Forexample, a selection period comprises decreasing or stopping removal ofcaprylate from the reaction mixture (e.g., by decreasing or stoppingextraction of caprylate from the reaction mixture). In an example, aselection period comprises decreasing or stopping removal of caprylatefrom the reaction mixture (e.g., by decreasing or stopping extraction ofcaprylate from the reaction mixture) for, for example, 0.5 hours to 720days, including all 0.1 hour values and ranges therebetween. In variousexamples, a selection period comprises decreasing or stopping removal ofcaprylate from the reaction mixture (e.g., by decreasing or stoppingextraction of caprylate from the reaction mixture) for 0.5 hours to 24hours, 0.5 hours to 120 hours, or 0.5 hours to 240 hours. In otherexamples, the selection period can be longer than 30 days. In variousexamples, a selection period is carried out until the concentration ofcaprylate(s) (e.g., undissociated caprylate (caprylic acid)) is 0.01% orgreater, 0.005% or greater, or 0.01% or greater by weight based on basedon the total weight of the reaction medium. In various examples, aselection period is carried out until the concentration of caprylate(s)(e.g., undissociated caprylate (caprylic acid)) is 0.1 g COD/L orgreater, 0.05 g COD/L or greater, or 0.1 gCOD/L or greater. Selectionperiod(s) can be carried out as part of an acclimation phase and/orsubsequent to an acclimation phase and/or as part of a production phaseand/or subsequent to a production phase.

A method can comprise a production phase (also referred to herein as aperiod). During a production phase caprylate products are formed in thereaction medium. In various examples, during a production phase then-caprylate productivity is at least 15 or at least 20 g chemical oxygendemand (COD)/L-d and/or the product ratio of n-caprylate to n-caproateof at least 10, at least 15, at least 20, or at least 25 g COD/g COD.

A product composition is formed by removal (e.g., by liquid extraction)of product compounds from the reaction mixture. Examples of liquidextraction are provided herein. A product composition comprises one ormore caprylate. In an example, a product composition comprises greaterthan 50% by weight caprylate(s) e.g., n-caprylic acid, n-caprylate or acombination thereof) based on the total weight of all the productcompounds (e.g., caprylate(s) and caproate(s)) in the productcomposition. In various other examples, a product composition comprisesgreater than 55% by weight, greater than 60% by weight, greater than65%, greater than 70%, greater than 80% by weight, greater than 90% byweight, greater than 95% by weight, or greater than 99% by weightcaprylate(s) (e.g., n-caprylic acid, n-caprylate or a combinationthereof) based on the weight of all of the product compounds (e.g.,caprylate(s) and caproate(s)) in the product composition. In an example,a product composition comprises 50-100% by weight caprylate(s) (e.g.,n-caprylic acid, n-caprylate or a combination thereof) based on theweight of all of the product compounds in the product composition. Theproduct composition can further comprise a solvent or mixture ofsolvents. For example, the solvent(s) are those used to extract theproduct compounds from the reaction mixture.

In various examples, a product composition comprises less than 20%, lessthan 10%, less than 5%, or less than 1% by weight caproate products(e.g., caproic acid, caproate, or combinations thereof) based on theweight of all of the product compounds in the product composition. Invarious examples, a product composition has a caprylate(s):caproate(s)weight ratio of 3:1 or greater, 4:1 or greater, 5:1 or greater, 10:1 orgreater, or 20:1 or greater. In an example, a product compositioncomprises no detectible caproate products. Caproate products can bedetected by methods known in the art. For example, caproate products aredetected by gravimetric methods, spectroscopic methods (e.g., nuclearmagnetic resonance (NMR) spectroscopic methods), or mass spectrometrymethods (e.g., gas or liquid chromatography/mass spectrometry methods).

The steps of the methods described in the various embodiments andexamples disclosed herein are sufficient to carry out the methods of thepresent disclosure. Thus, in an example, a method consists essentiallyof a combination of steps of one or more of the methods disclosedherein. In another example, a method consists of such steps.

In an aspect, the present invention provides systems for producingcaprylic acid. The systems comprise a continuous extraction system(e.g., an in-line continuous extraction system). Examples of systemsinclude, but are not limited to, anaerobic upflow bioreactors comprisinga continuous extraction system (e.g., an in-line continuous extractionsystem). The systems can carry out a method of the present disclosure.Examples of systems are provided herein.

A system for forming a product composition comprising caprylate productscomprises: a substrate (feed) source; a bioreactor in fluidcommunication with the substrate source, where the bioreactor includesan upflow anaerobic filter; and an in-line extraction system (alsoreferred to herein as a pertraction system) in fluid communication withthe bioreactor, where the in-line pertraction system includes: a forwardmembrane contactor, a backward membrane contactor; and an alkalineextraction solution source, where the in-line extraction (pertraction)system is configured to continuously recover hydrophobic, undissociatedmedium chain carboxylic acids from a bioreactor reaction medium from thebioreactor through the forward membrane contactor, and wherein themedium chain carboxylic acids are configured to be transferred acrossthe backward membrane contactor to an alkaline extractor solution fromthe alkaline extraction solution source. For example, the in-lineextraction (pertraction) system is configured for liquid-liquidextraction.

In an example, a system further comprises: a pH sensor connected to thebioreactor; and a controller in electronic communication with the pHsensor, where the controller is configured to maintain the bioreactorreaction medium at a particular pH. In an example, a system furthercomprising an acid addition pump in fluid communication with thebioreactor and in electronic communication with the controller. In anexample, the bioreactor includes an inverted funnel configured tocollect biogas. In an example, the in-line pertraction system includes aperistaltic pump.

The following examples are presented to illustrate the presentdisclosure. They are not intended to limiting in any matter.

EXAMPLE 1

This example provides a description of methods and systems of thepresent disclosure.

The operating conditions of an anaerobic upflow bioreactor was optimizedduring a period of 185 days to accomplish the goal of producingdesirable amounts of n-caprylate. We considerably increased then-caprylate productivity to 19.4 g chemical oxygen demand (COD)/L-d witha product ratio of n-caprylate to n-caproate of 11 g COD/g COD. Thisratio was even 25 g COD/g COD at an earlier operating period with alower productivity, resulting in a specificity of 96% when compared toall carboxylates. We accomplished this high n-caprylate productivity andspecificity by: 1) feeding a substrate with ethanol as the sole carbonsource or alternatively, a high ethanol-to-acetate ratio as the solecarbon source; 2) extracting the n-caprylate product from the bioreactorbroth; and 3) acclimating an efficient chain-elongating microbiome.Because syngas fermentation effluent consists of a high ratio of ethanolto acetate, these syngas fermentation products were coupled with chainelongation to increase n-caprylate product value.

Until now, mainly n-caproate has been produced with chain elongation. Weshow here, for the first time, that n-caprylate at high specificitiescan be produced as well. One of the requirements is a high substrateratio of ethanol to acetate, which is a characteristic of syngasfermentation effluent, opening up a new resource-recovery path towardthe production of sustainable and extractable fuels and chemicals. Forthis Example, ethanol as the electron donor was likely first producedfrom these gases by the microbiome.

Since the highest n-caprylate productivity was achieved with asynthetic, dilute ethanol and acetate substrate solution, the questionis where this particular substrate would come from in a sustainablesociety. Fortunately, waste-derived syngas-fermentation effluentprovides an emerging source of dilute ethanol and acetate for thesustainable production of MCCs. The syngas platform converts diversefeedstocks, including forestry residues and other relatively dry biomasswastes, into carbon monoxide- and hydrogen-rich producer gas, which werefer to here as syngas, via thermochemical processes. In addition,certain industrial off-gasses, such as those from steel mills, serve asvast sources of carbon monoxide and hydrogen gases as well. The emergingfield of syngas fermentation with anaerobic carboxytrophic clostridiahas been successful in converting these carbon monoxide-rich gases intodilute streams of ethanol and acetate. Others had already reported highethanol productivities of up to 10 g/L-h (500 g COD/L-d) andethanol-to-acetate ratios of up to 30 (based on COD). As a result,industrial-scale fermenters at steel plants are being designed and builtto convert syngas into ethanol via microbial fermentation.

Subsequent conversion of the dilute ethanol and acetate from syngasfermentation with chain elongation would be advantageous for severalreasons: 1) the value for C8 molecules is considerably higher than forC2 molecules on a weight basis; 2) conventional product recovery usingenergy-intensive distillation can be cost-prohibitive for recovery ofdilute ethanol, while hydrophobic MCC products can be extracted at alower expenditure of energy; and 3) without chain elongation, acetatereject from distillation would need to be treated in a wastewatertreatment plant. Since both syngas fermentation and chain elongation areanaerobic bioprocesses, they are complementary with similar temperatureand pH optimums and with growth nutrients that can be shared.

Here, we provided synthetic syngas-fermentation effluent with diluteethanol and acetate at a high substrate ratio of 15 (based on COD) to acontinuously fed bioreactor with a microbiome at a pH value of 5.5 andwith in-line product extraction. In addition, we performed batchexperiments to understand the effect of varying substrate concentrationsand ratios on microbiome performance. We achieved a high product ratioof n-caprylate to n-caproate by optimizing the operating conditions of abioreactor with in-line product recovery. Our results show that we canmainly produce n-caprylate from syngas-fermentation effluent with verylittle n-caproate as a co-product.

Experimental. Continuously fed bioreactor system. An upflow anaerobicfilter was used with constant bioreactor broth recycling through anin-line membrane liquid-liquid extraction (i.e. pertraction) system(FIG. 8). The bioreactor was a vertically oriented cylinder, which wasmade of Plexiglas®, with an inner diameter of 6 cm. The total volume was0.90 L, but Kaldnes K1 packing material (Evolution Aqua, Wigan, UnitedKingdom) was added, resulting in a working volume of 0.70 L. Thebioreactor was wrapped with tubing in which hot water from a heatingbath (VWR Scientific Model 1104, Radnor, Pa., USA) was recirculated fortemperature control, resulting in a constant temperature of 30±1° C.inside the bioreactor. A pH probe (Mettler 405-DPAS SC K85, Columbus,Ohio, USA) was mounted at the top of the bioreactor. Automated pHcontrol of the bioreactor broth was maintained with a controller (EutechInstruments alpha-pH800, Vernon Hills, Ill., USA) and a correspondingacid addition pump (Mityflex 913, Bradenton, Fla., USA). Hydrochloricacid (0.5 M) was added to the well-mixed feed and recycle inlet at thebase of the bioreactor. Fresh media containing ethanol and acetate wascontinuously fed from a refrigerated vessel (4° C.) into the base of thebioreactor using a peristaltic feed pump (Cole Parmer L/S DigitalEconomy Drive, Vernon Hills, Ill., USA) at average rates of ˜0.18 or0.44 L/d (hydraulic retention time [HRT]=3.9 or 1.6 d, respectively).The effluent continuously exited the bioreactor via an overflow lineconnected to the top of the bioreactor. The exit of the overflow linewas submerged within a secondary effluent reservoir. An inverted funnelwas used to collect the biogas within the bioreactor and was connectedto a flow meter (Ritter MGC-1, Bochum, Germany). In addition, agas-sample septum and a bubbler were placed in the gas collectionsystem. A sampling port for biomass samples was placed halfway up thevertically oriented bioreactor.

Pertraction system. Product extraction was accomplished with apertraction system similar to those used in previous reports. A forwardand a backward membrane contactor (1.4 m² each, Membrana Liqui-Cel2.5×8, X50 membrane, Charlotte, N.C., USA) were used for the bioreactorsetup (FIG. 8). A hydrophobic solvent was circulated continuously in thelumen of the hydrophobic hollow-fiber membrane modules as a selectivebarrier to extract primarily MCCs instead of SCC; the solvent consistedof mineral oil with 30 g/L tri-n-octylphosphine oxide (TOPO) (SigmaAldrich, St. Louis, Mo., USA). The stirred alkaline extraction solutionwas initially buffered with 0.3 M sodium borate and was maintained at pH9 with automated addition of 5 M sodium hydroxide using a controller(Eutech Instruments alpha-pH800, Vernon Hills, Ill., USA) and acorresponding base pump (Mityflex 913, Bradenton, Fla., USA). A constantbioreactor broth recycle flow of 130 L/d was maintained using aperistaltic pump (ColeParmer 7553-30, Vernon Hills, Ill., USA). Toprevent fouling or solids accumulation in the forward membranecontactor, bioreactor broth was drawn from the top of the anaerobicfilter and was then pumped through a custom-built, 1.6-mmstainless-steel strainer (Danco 88886, Shorewood, Ill., USA), a 65-μmfilter (McMaster-Carr 44205K21, Elmhurst, Ill., USA), and a subsequent5-μm filter (Pentek GS-6 SED/5, Upper Saddle River, N.J., USA).Peristaltic pumps (ColeParmer 7553-30, Vernon Hills, Ill., USA) providedcontinuous recycle flows of 7 and 43 L/d for the mineral oil solvent andalkaline extraction solution, respectively.

For abiotic pertraction experiments an aqueous solution of procuredsynthetic n-caproate was continuously fed to the abiotic upflowanaerobic filter after adjusting the pH to 5.5. The flow rate of thebioreactor broth recycle was varied to determine the effects of flowrate on mass transfer (but all other flow rates were held constant,including the mineral oil solvent, the alkaline extraction solution, andthe aqueous n-caproate feed solution). For the abiotic experiment weused a larger forward and backward membrane contactors than for thebioreactor experiment, but with identical hydrophobic hollow-fibermembranes (8.1 m² each, Membrana Liqui-Cel 4×13, X50 membrane,Charlotte, N.C., USA). We corrected for the difference in superficialvelocity.

Periods for the bioreactor study. This bioreactor was divided into twophases: I) a start-up phase with continuous feeding (with the productextraction system on or off); and II) a high n-caprylate productivityphase (with continuous feeding and the product extraction system on).Each phase was then divided into several distinct operating periods.From period to period, several operating parameters were varied,including the: organic loading rate (OLR); HRT; bioreactor pH; andoperation with or without product extraction (Table 1). Each operatingperiod was operated for at least five HRT periods, and averagebioreactor loading rates and concentrations were reported.

Table 1. Operating conditions and average bioreactor brothconcentrations. Average substrate and product concentrations in thebioreactor broth are reported for each operating period with theircorresponding operating conditions. Detection limits were approximately0.05 g COD/L (0.5 mM) for ethanol and approximately 0.02 g COD/L (˜0.1mM) for other carboxylates. B.D.: below detection. Uncertainty isrepresented by 95% confidence intervals.

TABLE 1 Operating conditions Average bioreactor broth concentrationsPhase Period Pertraction Start~End Bioreactor [Ethanol] [n-Caprylate][n-Caproate] [n-Butyrate] [Acetate] [Other SCC] # # +/− d pH g COD/L I1 +  0~15 5.8 ± 0.6 1.01 ± 1.11 B.D. 0.80 ± 0.50 0.13 ± 0.08 0.64 ± 0.120.03 ± 0.03 2 + 15~54 5.6 ± 0.2 0.10 ± 0.01 B.D. 0.19 ± 0.07 0.11 ± 0.050.57 ± 0.09 0.02 ± 0.01 3 + 54~64 5.4 ± 0.1 0.10 ± 0.01 0.05 ± 0.12 0.03± 0.09 0.01 ± 0.01 0.39 ± 0.10 0.01 ± 0.03 4 − 64~80 5.5 ± 0.1 0.04 ±0.04 0.21 ± 0.14 0.23 ± 0.12 0.03 ± 0.03 0.61 ± 0.08 0.05 ± 0.03 5 −80~98 5.3 ± 0.1 0.25 ± 0.24 0.76 ± 0.16 1.03 ± 0.33 0.60 ± 0.24 0.75 ±0.10 0.15 ± 0.03 II 6 +  98~128 5.2 ± 0.1 0.03 ± 0.05 0.03 ± 0.02 0.07 ±0.02 0.08 ± 0.02 0.22 ± 0.05 0.01 ± 0.01 7 + 128~142 5.1 ± 0.1 0.02 ±0.05 0.04 ± 0.02 0.09 ± 0.02 0.07 ± 0.02 0.13 ± 0.06 0.03 ± 0.02 8 +142~155 5.0 ± 0.1 0.99 ± 0.20 0.09 ± 0.02 0.06 ± 0.04 0.00 ± 0.00 0.01 ±0.02 0.06 ± 0.03 9 + 155~163 5.1 ± 0.1 0.49 ± 0.24 0.18 ± 0.03 0.08 ±0.03 0.05 ± 0.05 0.09 ± 0.04 0.05 ± 0.03 10 + 163~174 5.1 ± 0.1 8.67 ±2.12 0.34 ± 0.08 0.28 ± 0.06 0.11 ± 0.03 0.13 ± 0.02 0.09 ± 0.06 11 +174~186 5.2 ± 0.1 27.33 ± 3.01  0.69 ± 0.10 1.12 ± 0.11 1.09 ± 0.38 0.57± 0.12 0.04 ± 0.02

During Period 1 in Phase I (Days 0-15), pre-washed inoculum was added tothe upflow anaerobic filter after which a continuous feeding strategywas initiated. The substrate ratio of ethanol to acetate was 6 (g COD/gCOD), the HRT was 4.2 days, and the total OLR was 2.1 g COD/L-d (Table2). The bioreactor was re-inoculated on Day 15 of Period 2 in Phase I(Days 15-54) with pre-washed inoculum, and then again on Day 54 ofPeriod 3 of Phase I (Days 54-64) with non-washed inoculum. This lastinoculation carried some carboxylates from the inoculation bioreactor.During Period 4 of Phase I (Days 64-80), the product extraction wasturned off. This action was taken to encourage an increase inconcentrations of undissociated medium-chain carboxylic acids (MCCAs),such as undissociated n-caproic acid and n-caprylic acid, in thebioreactor broth. On Day 80 of Period 5 in Phase I (Days 80-98), weincreased the substrate ratio of ethanol to acetate from 6 to 15 g COD/gCOD. In addition, we increased the OLR from 1.8 to 3.8 g COD/L-d (Table2). Day 98 represented the start of Phase II (Period 6; Days 98-128), byswitching on the product extraction system. Next, we increased the OLRsin a step-wise fashion during 4 out of the next 5 periods in Phase II.In Period 7 (Days 128-142) and 8 (Days 142-155), the OLR was increasedfrom 3.7 to 6.3 and to 15.0 COD/L-d without changing the HRT. On day 155of Period 9 in Phase II (Days 155-163), we shortened the HRT from 3.3 to1.6 days by increasing the flow rate of the growth medium. However, wemaintained a similar OLR by reducing the ethanol and acetateconcentrations. By increasing the flow rate on Day 155, the averageyeast-extract loading rate increased from 0.4±0.1 g COD/L-d (for thefirst eight periods at an average HRT of 3.9±0.1 d) to 1.1±0.1 g COD/L-d(for the last three periods at an average HRT of 1.5±0.1 d). Furtherincreases in the OLR were made on Day 163 (Period 10; Phase II; Days163-174) to 34.7 g COD/L-d and on Day 174 (Period 11; Phase II; Days174-186) to 63.8 g COD/L-d (Table 2).

Table 2. Operating conditions and average loading rates and carboxylateproductivities. Average productivities of medium-chain carboxylates(MCCs) (e.g. n-caprylate, n-caproate) and short-chain carboxylates(SCCs) are reported for each operating period with their correspondingoperating conditions. These total productivities were calculated as thesum of average bioreactor effluent production rates plus averagetransfer rates via pertraction for each operating period, normalized tothe bioreactor working volume. Acetate was continuously fed to thebioreactor, so negative production rates indicate net consumption ofacetate. Uncertainty is represented by 95% confidence intervals. Totalorganic loading rates (OLRs) include loading from ethanol, acetate, andyeast extract. For most of the experiment, to vary the ethanol and totalorganic loading rates, the concentrations of ethanol and acetate in thebasal medium were changed (instead of the HRT). The substrate ratio ofethanol to acetate was approximately 6 g COD/g COD until it wasincreased to 15 g COD/g COD on Day 80 of Phase I, Period 5. For eachoperating period, the concentrations of ethanol and total organics (gCOD/L) in the continuously fed basal medium can be calculated bymultiplying the reported average ethanol and total organic loading rates(g COD/L-d) by the corresponding average HRT (d). The yeast extractconcentration in the media was consistently 1.6 g COD/L (1.25 g/L), andthe corresponding yeast extract loading rate was approximately 0.4±0.1 gCOD/L-d throughout the first 8 operating periods (HRT=3.9±0.1 d). InPeriod 9, the feed flow rate was increased, which decreased the HRT, andthe yeast extract loading rate was consequently increased to 1.1±0.1 gCOD/L-d (HRT=1.5±0.1 d). No considerable changes were observed in then-caprylate or the total MCC productivities between Period 8 and Period9. Uncertainty is represented by 95% confidence intervals.

TABLE 2 Operating conditions Loading rates Average carboxylateproductvities Phase Period Pertraction HRT Total OLR Ethanol n-Caprylaten-Caproate n-Butyrate Net Acetate Other SCC # # +/− d g COD/L-d gCOD/L-d I 1 + 4.2 ± 0.6 2.1 ± 0.6 1.4 ± 0.4 0.0 ± 0.0 0.5 ± 0.3 0.0 ±0.0 −0.1 ± 0.0 0.0 ± 0.0 2 + 4.8 ± 0.6 1.8 ± 0.1 1.2 ± 0.1 0.0 ± 0.0 0.3± 0.1 0.3 ± 0.2 −0.1 ± 0.0 0.3 ± 0.1 3 + 3.8 ± 0.3 1.7 ± 0.4 1.2 ± 0.20.0 ± 0.0 0.6 ± 0.3 0.0 ± 0.0 −0.1 ± 0.0 0.0 ± 0.0 4 − 4.5 ± 0.9 1.8 ±0.3 1.2 ± 0.2 0.0 ± 0.0 0.1 ± 0.0 0.0 ± 0.0 −0.1 ± 0.0 0.0 ± 0.0 5 − 4.4± 0.2 3.8 ± 0.4 3.2 ± 0.3 0.2 ± 0.0 0.2 ± 0.1 0.1 ± 0.1  0.0 ± 0.0 0.0 ±0.0 II 6 + 3.7 ± 0.2 3.7 ± 0.4 3.1 ± 0.3 0.8 ± 0.1 1.2 ± 0.2 0.3 ± 0.1−0.2 ± 0.0 0.0 ± 0.0 7 + 3.8 ± 0.4 6.3 ± 0.4 5.6 ± 0.4 2.3 ± 0.4 2.2 ±0.2 0.0 ± 0.0 −0.3 ± 0.0 0.1 ± 0.1 8 + 3.3 ± 0.3 15.0 ± 2.9  13.7 ± 2.6 10.6 ± 0.3  0.4 ± 0.1 0.0 ± 0.0 −0.9 ± 0.2 0.0 ± 0.0 9 + 1.6 ± 0.1 13.7± 1.8  12.2 ± 1.6  11.2 ± 1.5  1.7 ± 0.3 0.0 ± 0.0 −0.8 ± 0.1 0.3 ± 0.210 + 1.5 ± 0.1 34.7 ± 2.7  31.8 ± 2.5  19.4 ± 1.1  1.7 ± 0.3 0.1 ± 0.0−2.0 ± 0.2 0.1 ± 0.0 11 + 1.5 ± 0.0 63.8 ± 6.7  59.1 ± 6.2  13.2 ± 0.8 7.5 ± 0.3 0.7 ± 0.2 −3.6 ± 0.4 0.0 ± 0.0

Growth medium and inoculum. The modified basal medium was describedpreviously, and it contained nutrients, yeast extract (1.25 g/L, 1.6 gCOD/L), and sodium carbonate (0.032 g/L), but no gaseous carbon dioxide.Ethanol and acetate were added to the basal medium at a fixed substrateratio of 6 g COD/g COD (4 mol/mol) until Day 79, and was then increasedto 15 g COD/g COD (10 mol/mol) on Day 80 (Period 5). For each operatingperiod, the substrate concentrations of ethanol and acetate were variedto achieve desired loading rates (Table 2). The medium pH was adjustedwith 5 M sodium hydroxide to achieve an appropriate pH in the bioreactor(Table 1). The inoculum was derived from a well-characterized reactormicrobiome that was fed ethanol-rich yeast fermentation beer. Thisreactor microbiome had been fed semi-continuously (once every two days)throughout an operating period of more than three years at the time ofinoculation. For pre-washed inoculation, the inoculum was triple-washedin basal media, and ˜100 mL of this inoculum was added to thecontinuously fed bioreactor. For the non-washed inoculation, 100 mL ofinoculum was added.

Batch reactor microbiome experiments. Batch reactor microbiomeexperiments were conducted in 160-mL glass serum bottles to which 80 mLof basal medium was added. The initial concentrations and substrateratios of ethanol and acetate in the basal medium were varied. MESbuffer was also added to this media at concentrations that wereequimolar to the initial ethanol concentrations, and the initial pH wasadjusted to 5.4 with 5 M sodium hydroxide. Inoculum was prepared asdescribed previously, and ˜4 mL of pre-washed inoculum was added to 80mL volume (5% inoculum, v/v). The batch reactor microbiomes were then:sparged with nitrogen gas; capped with butyl rubber stoppers; sealed andcrimped with aluminum caps; inverted; and incubated without shaking at30° C. These serum bottles were then mixed well and sampled after 12days, and liquid samples (pH 5.4±0.1) were collected in 2-mL Eppendorftubes for determination of the concentrations of ethanol andcarboxylates. Each treatment was conducted in triplicate batch bottles,and all data reported reflect the average values from these triplicates.

Calculations. We use g COD for our results instead of g product tocompare results and to ascertain the balance between substrate andproduct. The conversion factors for 1 g COD to g product are: 0.48(ethanol); 0.92 (acetate); 0.65 (propionate); 0.54 (n-butyrate); 0.49(n-valerate); 0.45 (n-caproate); 0.42 (n-heptanoate); and 0.41(n-caprylate). Carboxylate productivities were calculated as averagevalues for each operating period. Herein, the average bioreactoreffluent production rate (g COD/d) plus the average transfer rate viaproduct extraction (g COD/d) were summed to yield the total productionrate (g COD/d). Effluent production rates were calculated as the averagebioreactor broth concentration divided by the average HRT for eachperiod. Average transfer rates were calculated by first plotting theincreasing amounts of individual carboxylates in the alkaline extractionsolution against time. Least squares methods were then used to determinethe slope and the sample standard deviation (LINEST function, MicrosoftExcel). We divided the production rates by the working bioreactor volumeto determine the total productivities (g COD/L-d). All concentrations,rates, and yields were converted to a g COD basis. Feed flow rates weredetermined volumetrically; effluent rates were determinedgravimetrically. Uncertainty was represented by 95% confidenceintervals: the standard error was first calculated as the quotient ofthe sample standard deviation divided by the square root of the numberof samples; then, the standard error was multiplied by a t-valuecorresponding to the degrees of freedom (based on the number ofsamples). Uncertainty was propagated through calculations, and 95%confidence intervals were included with reported data (e.g.productivities).

Liquid and gas analysis. Liquid samples (1.5 mL) were collected from thecontinuously fed bioreactor and the alkaline extraction solution everyother day or daily. Bioreactor broth samples were collected from thebroth recycle line between the 5-μm filter and the forward membranecontactor. Alkaline extraction solution samples were collected from thewell-mixed reservoir (˜3 L). Concentrations of carboxylates and ethanolwere determined with separate gas chromatography (GC) systems. Theconcentrations of methane, carbon dioxide, and hydrogen gases (>2000ppm) were measured using a GC system. Furthermore, the concentration ofhydrogen gas (<2000 ppm) was determined using a reduction gas detector(RGD) (Trace Analytical RGD, Menlo Park, Calif., USA). The RGD inlet wasconnected to a packed column (Restek, ShinCarbon ST 80/100, Bellefonte,Pa., USA) for peak separation, which was installed in a GC system (GowMac 580, Bethlehem, Pa., USA).

Biomass samples, DNA extraction, PCR, sequencing, and microbialcommunity analysis. Biomass samples were taken from the bioreactor brothat 16 time points throughout the experimental period, as well as onesample from the inoculum. The bioreactor broth was thoroughly mixed byquickly withdrawing and refilling a 60 mL syringe ten times. During thissampling, settled flocculent biomass was resuspended. The sample wascollected in 2-mL Eppendorf tubes. These 2-mL samples were thencentrifuged at 16,873×g for 4 min and the supernatants were discarded.Concentrations of wet solids in these pelleted biomass samples rangedfrom 23 to 76 mg/L. These pelleted biomass samples were stored at −80°C. until further processing.

Genomic DNA was extracted using the PowerSoil DNA isolation kit (MO BIOLaboratories Inc., Carlsbad, Calif.). Modifications to the protocolinclude utilization of custom bead tubes containing a mixture of 300 mgof 0.1-mm diameter and 100 mg of 0.5-mm diameter silica/zirconia beads,and physical cell lysis with bead-beating at 3450 oscillations/min for45 s. The DNA amplification protocol was described previously with thefollowing exceptions: 1) Mag-Bind RxnPure Plus magnetic beads solution(Omega Biotek, Norcross, Ga., USA) were used instead of Mag-Bind E-ZPure; 2) only 20 ng DNA per sample were pooled instead of 100 ng. QIITA(qiita.microbio.me) was used for initial processing of the sequencingdata. The sortmerna method was used to bin sequences into operationaltaxonomic units (OTUs) at 97% identity. Taxonomy was assigned forrepresentative sequences selected for each OTU using the Greengenesv13.8 database from August 2013. The remaining analyses were performedin QIIME v1.9. Singleton OTUs were removed from the dataset.

Community analysis, including beta diversity and unconstrainedordination, was performed as described previously with the followingexceptions: 1) the alpha diversity was calculated using the Shannondiversity index rather than Chaol; 2) the Pearson correlationcoefficient was calculated for samples from Phase III with the functionscor and cor.test in the R stats package. At a significance level ofp<0.05 and n=11, the relative abundance of an OTU would be positivelycorrelated with n-caproate productivity if the Pearson r was greaterthan 0.602. Heat maps were created to represent OTU relative abundancesvia the gplots package in R.

Results and Discussion. We achieved the highest n-caprylate productivityand specificity ever reported. During a period of more than 180 days weoperated an upflow anaerobic filter with and without product extractionand with a continuous feed of synthetic ethanol and acetate. We achieveda maximum average MCC productivity of 21.1 g COD/L-d in this Exampleduring Period 10 (FIG. 1). The corresponding n-caprylate productivitywas 19.4 g COD/L-d (Table 2), which is more than four times the highestn-caprylate productivity (4.4 g COD/L-d) reported previously, which wasachieved without product extraction and at a much higher OLR (FIG. 2).Importantly, the product ratio of n-caprylate to n-caproate was 11(based on COD) during this operating period of 11 days in Period 10(Table 1) at an HRT of 1.5 d (Table 2). During an earlier period at alower MCC productivity (Period 8), we achieved a product ratio ofn-caprylate to n-caproate of 25 (FIGS. 1-2). Both these product ratiosare considerably higher than what had been observed. Previously, themaximum reported value was 1.47, but at very low productivities. Thus,this is the first Example that has reported mainly n-caprylate at a veryhigh product specificity (product vs. other carboxylate products) of 96%(Period 8) and 91% (Period 10), which included all SCC and MCC productsfrom this operating period (on a COD basis).

Even though the MCC productivity was high during the period with amaximum n-caprylate productivity (Period 10), we were over feeding thebioreactor slightly with a total OLR of 34.7 g COD/L-d (FIG. 1). Thisresulted not only in the observed decrease in the product ratio ofn-caprylate to n-caproate from 25 to 11 (Period 8 to 10), but alsoresulted in a decrease in the COD conversion efficiencies to MCCs from73% to 61% (n-caprylate plus n-caproate in COD/total OLR in COD; Table3), respectively. The COD conversion efficiencies to MCCs based onethanol COD decreased from 80% to 67% during this operating period(Table 3). A further increase in the OLR to 63.8 g COD/L-d during Period11 resulted in decreases in the n-caprylate productivity, then-caprylate-to-n-caproate ratio (1.7), and the COD conversionefficiency, while the total MCC productivity stagnated (FIG. 1). Theover-feeding conditions during Period 10-11 led to increases in theconcentrations of: 1) carboxylates (FIG. 3A) ethanol (FIG. 3B) in thebroth of the bioreactor; and 2) hydrogen and methane in the biogas ofthe bioreactor (FIG. 9). The concentrations of hydrogen and methane onlyincreased considerably during Period 11 to reach concentrations above3,000 and 30,000 ppm, respectively (FIG. 9). This occurred when a largeexcess of reducing equivalents became available. A considerable fractionof the produced hydrogen was converted to methane via hydrogenotrophicmethanogens at a pH of 5.2 with carbon dioxide being the limitingsubstrate for these methanogens. Clearly, it is important to not overfeed the bioreactor when the objective is to achieve a high productratio of n-caprylate to n-caproate.

A high substrate ratio of ethanol to acetate is needed to obtain a highproduct ratio of n-caprylate to n-caproate. During Phase II when highn-caprylate productivities were achieved, the substrate ratio of ethanolto acetate was 15. We had increased this ratio from 6 to 15 in Period 5when product extraction was off. This change was made in tandem with anincrease in the OLR from 1.8 to 3.8 g COD/L-d. These changes led to anincrease in the n-caprylate-to-n-caproate ratio to 0.5 (FIG. 1). Anethanol-to-acetate ratio of 4.4 g COD/g COD without product extractionwas previously used and achieved the highest n-caprylate productivitybefore this work, but with a relatively low n-caprylate-to-n-caproateratio. The lower ethanol-to-acetate ratio may explain the considerablylower product ratio of 0.04 compared to 0.5 g COD/g COD, which weachieved without product extraction.

It was previously known with pure cultures of C. kluyveri in batchexperiments that increasing the substrate ratios of ethanol to acetateled to increased product ratios of n-caproate (C6) to n-butyrate (C4)(FIG. 10). Production of n-caprylate was not seen. For example, when theconcentration of ethanol was increased with a fixed acetateconcentration, the product ratio of n-caproate to n-butyrate and then-caproate productivity increased until the ethanol-to-acetate ratio was6 (based on COD) and the ethanol concentration of 44 g COD/L (460 mM)became inhibiting (FIG. 10C). In a recent review, a thermodynamic modelshowed that both a higher substrate ratio of ethanol to acetate and ahigher product ratio of n-caproate to n-butyrate ratio would beenergetically advantageous for C. kluyveri. In relation to syngasfermentation, the relatively high ethanol-to-acetate ratios up to 30 (gCOD/g COD) in syngas-fermentation effluent are an important advantagefor chain elongation to a longer product. Use of this syngasfermentation product in reactor systems produced n-butyrate andn-caproate but n-caprylate production was not seen²⁸.

Even though production of n-caprylate has not yet been reported withpure cultures of C. kluyveri, it has so with microbiomes. We, therefore,performed a batch experiment with microbiomes at a mildly acid pH toascertain whether substrate ratios would have an effect on productratios with n-caprylate. In general, a higher ethanol-to-acetate ratio,indeed, resulted in a higher n-caprylate-to-n-caproate ratio (FIG. 4).For the fixed starting ethanol concentration of 9.6 g COD/L (100 mM),this was true in the batch experiments for the entire range of substrateratios (yellow circles in FIG. 4 and FIG. 11A). Two other observationsfrom this batch experiment are pertinent, though: 1) a higher initialethanol concentration lowers the n-caprylate-to-n-caproate ratio foreach ethanol-to-acetate ratio (FIG. 4). For the fixed ethanol-to-acetateratio of 13.5 g COD/g COD this is most clear with a considerably lowern-caprylate-to-n-caproate ratio throughout the entire range ofincreasing initial ethanol concentrations (third column from left inFIG. 4 and FIG. 11B); and 2) ethanol is inhibiting n-caprylateproduction and not acetate (FIG. 4). For the relatively low, fixedacetate concentration of ˜0.7 g COD/L (˜10 mM) with increasing initialethanol concentrations, the product ratios and the n-caprylateconcentration first increased, but then decreased at the two highestinitial ethanol concentrations (FIG. 11C). In fact, we observed aconsiderable inhibition at an initial ethanol concentration of 28.8 gCOD/L (300 mM) ethanol without accumulation of the possible inhibitingundissociated MCCs (FIG. 11B-C). From this work it is clear that formicrobiomes a positive correlation exists between the ethanol-to-acetateratio and the n-caprylate-to-n-caproate ratio, but that theconcentration of ethanol in the bioreactor should be maintained belowinhibiting conditions.

Product extraction is needed to obtain high product ratios ofn-caprylate to n-caproate. The highest n-caprylate-to-n-caproate ratiothat we achieved with microbiomes in our batch experiments withoutextraction was 2.7 g COD/g COD with an ethanol-to-acetate ratio of 13.5.Because we used similar conditions: the same inoculum, a close substrateratio of 15, and a mildly acidic pH, the much higher achieved substrateratio of 25 during Period 8 with our continuous anaerobic bioreactor canonly be explained by product extraction. Accordingly, we did observe anaverage n-caprylate productivity increase from 0.2 to 0.8 COD/L-d fromPeriod 5 to Period 6 when product extraction was started on Day 98without any other operating changes (Table 2).

To understand how product extraction can achieve such as large increasein product ratio, we should first discuss thermodynamics and productinhibition. A recently published thermodynamic model shows that a higherproduct ratio of n-caproate to n-butyrate is energetically favorable forC. kluyveri by releasing more ATP. In general, the longer the MCC thatis produced, the more reduced the chemical is, and the more ATP isreleased, which is advantageous for chain-elongating bacteria. However,thus far, the n-caprylate-to-n-caproate ratio has been low, whilen-capricate (C10) production has not been demonstrated. The considerablyhigher product inhibition from the longer MCCs in bioprocess systems canexplain this, because a strong correlation exists between the length ofthe chain and its toxicity. The pH value in the bioreactor broth playsan important role because the undissociated MCCAs inhibit microbialactivity with mildly acidic pH values for the pKa (4.88 for n-caproateand n-caprylate). The undissociated MCCAs (e.g. n-caproic acid,n-caprylic acid) are hydrophobic and their hydrophobicity increases forMCCAs with longer carbon chains. These MCCAs can, therefore, penetratethe hydrophobic lipid membranes of microbial cells and even damagecytoplasmatic structures. Previous work found such damage in entericbacteria. However, the membrane integrities remained intact, pointingtoward exhaustion from expelling protons to maintain a neutral pH in thecytoplasm as the mechanism of toxicity for MCCAs.

Researchers have used two different approaches to maintain lowconcentrations of undissociated MCCAs in the bioreactor broth with theoverarching goal to overcome product inhibition and to increase MCCproductivities. These two approaches from different operating conditionsare: 1) a neutral pH value of 6.5-7.5 and relatively short HRTs; and 2)a mildly acidic pH value of 5.0-5.5 and in-line product extraction.Here, we used the second approach. When reactor microbiomes are used tochain elongate, it is important to completely inhibit acetoclasticmethanogens to prevent acetate conversion into methane. We accomplishedthis by operating the bioprocess at a mildly acidic pH conditions. Inaddition to inhibiting methanogens, the mildly acidic pH conditionsensure a sufficient chemical gradient for product extraction from thebioreactor broth. Product extraction via pertraction is considerablymore efficient with longer carboxylates that have a ˜10× lower maximumsolubility concentration in their undissociated form for each 2 carbonsthat are added. This results in faster extraction rates for n-caprylatecompared to n-caproate and a selective pressure, and likely added to theexplanation of why our continuous bioreactor with in-line extractionachieved such superior n-caprylate productivities and selectivities.

Before the slight over feeding conditions during Period 8, we observedMCC extraction efficiencies of 99.7% and 95.5% for n-caprylate andn-caproate, respectively. However, these efficiencies decreased to 98.9%and 89.6%, respectively, during Period 10 and then 96.6% and 89.4%,respectively, during Period 11 when the OLRs were increased (Table 3).The resulting decrease in efficiency and the ability of microbiomes toachieve considerably higher MCC productivities with ethanol and acetateas a substrate than in this Example, indicates that in-line productextraction was limiting the production rates of our bioreactor. Sincethe biological chain elongation rates were higher than the extractionrates, MCCs accumulated in the bioreactor broth (Period 10-11 in FIG. 3and Table 2). Due to the mild acidic conditions of our bioreactor(pH=5.2), undissociated n-caprylic acid and n-caproic acidconcentrations were relatively high (pKa=4.88), possibly reachinginhibiting concentrations. Together with high concentrations of ethanolit explains why the MCC productivity stagnated in Period 11 (FIG. 1). Ina separate abiotic experiment, we observed that the flow rate of thebroth recycle (and not of the mineral oil solvent nor of the alkalineextraction solution) was directly proportional to the overall masstransfer coefficient of n-caproic acid (FIG. 12). Therefore, weincreased the MCC extraction rates, and thus the MCC productivities andthe n-caprylate specificities, in our Example by increasing therecycling flow of the bioreactor broth through the forward membranecontactor (FIG. 8).

The achieved maximum concentrations of n-caprylic acid and ethanol wereinhibiting. During Period 5 without product extraction, we observedmaximum average undissociated n-caproic acid and n-caprylic acidconcentrations of 0.27 g COD/L (1.1 mM) and 0.21 g COD/L (0.6 mM),respectively (FIG. 3A). These increasing concentrations of undissociatedcarboxylic acids resulted in the sudden increase in residual ethanolconcentrations to ˜0.5 g COD/L (5 mM) on Day 92 (FIG. 3B). Next,switching on the product extraction system on Day 98 in Period 6instantly removed the accumulated MCCs, resulting in an almost immediaterelief of the residual ethanol concentrations in the bioreactor (FIG.3A). Clearly, the high concentrations of undissociated carboxylic acidscaused microbial inhibition.

The maximum concentration of undissociated n-caproic acid of 1.1 mMduring Period 5 is considerably lower than the maximum concentrationsthat other studies have observed with microbiomes. For example, in aprevious report a maximum undissociated n-caproic acid concentration of10.5 mM in the bioreactor broth with ethanol as the electron donor,which is 11% of the solubility limit of undissociated n-caproic acid (93mM) was previously reported. At the same time, the maximum concentrationof undissociated n-caprylic acid of 0.63 mM in our bioreactor brothduring Period 11 is 13% of the solubility limit (4.7 mM) and isconsiderably higher than reported by previous studies (FIG. 13). Fromthis comparative analysis, we postulate that accumulated undissociatedn-caprylic acid was inhibiting our microbiome rather than n-caproicacid. This inhibition of undissociated n-caprylic acid was used as anecological tool to enrich for n-caprylate-producing bacteria in themicrobiome during Phase I. We had switched off the extraction on purposeto accumulate MCCAs during Period 4 after which, for the first time, then-caprylate-to-n-caproate ratio increased (FIG. 3A). Predominantlychain-elongating bacteria survived such high concentrations of MCCAs.

The ethanol concentration in the bioreactor broth increased to anultimate concentration of 33 g COD/L (350 mM) during Periods 10-11 (FIG.3B), but did not reach 44 g COD/L (460 mM), which had previously beenobserved to be inhibitory in pure culture studies of the type strain C.kluyveri. Possibly inhibiting ethanol concentrations need to be takeninto consideration because 33 and 44 g COD/L are considerably lower thanthe maximum ethanol concentration of ˜100 g COD/L (1 M) insyngas-fermentation effluent. However, an efficient chain elongationsystem can maintain a very low ethanol concentration in the bioreactorbroth (Periods 6-7) (FIG. 3B) or non-inhibiting ethanol concentrationsfor long operating periods⁹. Our batch experiments with microbiomes atan initial ethanol concentration of 28.8 g COD/L (300 mM) indicate thatthe average residual ethanol concentration of 27.3 g COD/L (284 mM)during Period 11 would have likely caused substrate inhibition. From ourresults with batch and bioreactor experiments it is, therefore, apparentthat both high undissociated MCCA concentrations and a residual ethanolconcentration of ˜300 mM will inhibit the microbial processes. Likely,some interaction between these different inhibitions will be present.

Microbiome analysis showed a surprising absence of the type strain C.kluyveri, We also investigated the microbiome dynamics during theoperating period. We observed 1634 operational taxonomic units (OTUs)from high-quality sequence reads with 48 of these OTUs exceeding 1% ofthe relative abundance in one or more reactor microbiome samples duringthe entire operating period (FIG. 14). In addition, these 48 OTUsaccounted for 88.1%-96.0% of the total high-quality sequence reads foreach sample. A total of 36 OTUs exceeded 1% of the relative abundance inat least one sample for Phase II during which we observed highn-caprylate productivities. These 36 OTUs were hierarchically rankedbased on both the average relative abundance and the abundance profilethroughout Phase II. This resulted in the highest abundant OTUs at thebottom of the heat map (FIG. 5). OTUs for Acinetobacter spp. and aRhodocyclaceae K82 spp. were predominant during Phase II. Between Days140-150 during Periods 7-8, the relative abundance of the Acinetobacterspp. OTU decreased. On the other hand, the relative abundance of theRhodocyclaceae K82 spp. OUT increased during Periods 8-10 with thehighest MCC productivities of this Example. Next, the relative abundanceof Rhodocyclaceae K82 spp. decreased during the over-loading conditionsof Period 11. We did not find a statistical correlation between theabundances for the OTUs of Acinetobacter spp. and of Rhodocyclaceae K82spp. and the n-caprylate productivities (p>0.05). However, we did findsuch correlations for five OTUs with a much lower abundance in themicrobiome (FIG. 5), including: 1) Desulfosporosinus meridiei (p=0.01);2) Oscillospira spp. (p=0.02); 3) Burkholderia spp. (p=0.02); and 4-5)unknown Ruminococcaceae (p=0.002; p=0.04). While the type strain C.kluyveri is known to elongate acetate into n-caproate with ethanol as anelectron donor, the highest relative abundant Clostridium OTU found inthis Example was less than two percent during Phase I. Thus, the absenceof C. kluyveri during Phase II, the lack of any pure-culture studiesthat reported n-caprylate as a product, and an optimum pH of 6.5-7.5,indicates that we have a different type strain for chain elongation ton-caprylate at mildly acidic conditions.

The number of OTUs within the community and their relative proportionsdid not vary considerably during the operating period (FIG. 15). Thebeta diversity, or dissimilarity of OTU composition between samples,showed a chronological path from earlier samples (lighter circles) tolater samples (darker circles) for Phase II with a high n-caprylateproductivity (FIG. 6A) and for the entire operating period including theinoculum (FIG. 16). The final sample from Day 186 (Period 11) was themost dissimilar sample during Phase II (darkest circle to the far rightin FIG. 6A). To ascertain whether the operating conditions,environmental parameters, or functional performance affected themicrobiome dissimilarity during Phase II with a high n-caprylateproductivity, we performed constrained ordination. We found twoparameters that explained 88% of the variation in the beta diversityduring Phase II: 1) residual ethanol concentrations in the bioreactorbroth (an environmental parameter); and 2) hydraulic retention time (andoperating condition) (FIG. 6B), while the sample day number was not asignificant (p>0.1) predictor of dissimilarity when considered in themodel along with these two parameters. The presence of residual ethanolis, thus, important in shaping the microbiome, and clearly we hadreached microbial inhibition with residual ethanol concentrations near28 g COD/L (˜300 mM) during Period 11 (FIG. 3B). In addition, weobserved in Phase I that the n-caprylate concentration can be inhibitingthe microbiome, however, we did not have enough samples for astatistically meaningful constrained ordination analysis. Finally, ourwork shows that the hydraulic retention time is an important parameterto shape the microbiome in our upflow anaerobic filter.

Conclusions. With a synthetic substrate of ethanol and acetate, whichmimicked syngas fermentation effluent, we observed a sustainedn-caprylate production via chain elongation with a reactor microbiome.The maximum n-caprylate productivity was 19.4 g COD/L-d at a productratio of n-caprylate to n-caproate of 11 g COD/g COD. At a lowerproductivity, this ratio was even 25 g COD/g COD, resulting in aspecificity of 96%. We obtained these results by combining: 1) a highsubstrate ratio of ethanol and acetate (15 g COD/g COD); 2) in-lineproduct extraction; and 3) the selection of an acclimated microbiomeform a sustained chain-elongating bioreactor. To our surprise, the typestrain C. kluyveri was absent from the reactor microbiome. We found thatboth high residual concentrations of undissociated n-caprylic acid (0.62mM) and ethanol (300 mM) inhibit microbial activity. In addition,residual ethanol concentrations affected the community structure of themicrobiome during a period of high n-caprylate productivity. Syngasfermentation effluent represents a renewable source of ethanol andacetate at high substrate ratios for chain elongation. Upgrading the C2molecules into predominantly C8 molecules by chain elongation representsa pathway to increase product value and reduce the energetic cost ofproduct extraction from a biotechnology platform that includes syngasfermentation. Little to no methane is produced using this system

EXAMPLE 2

This example provides a description of methods and systems of thepresent disclosure.

A carboxylate bioreactor utilizing reverse beta oxidation has been shownin the past to produce significant levels of caproate and high levels ofcaprylate compared to other studies. The purpose of this Example is toexamine the effects of influent molar ethanol to acetate ratios on theperformance of an anaerobic bioreactor, in particular, the caprylate,caproate, and butyrate production rates. The bioreactor was initiallyfed media with an ethanol to acetate molar ratio of 10:1, 10:0 (onlyethanol), 9:1, 8:2, 7:3, and 6:4. The reactor was operated for a periodof about two weeks. The results suggest that higher ethanol to acetateratios result in higher caprylate conversion efficiencies. This Examplealso demonstrated, for the first time, chain elongation in acontinuously fed reactor using only ethanol as the substrate.

The goal of the Example is to experimentally examine the effects of themolar feed ratio of ethanol to acetate on the production of medium chaincarboxylates in an anaerobic bioreactor more closely than has been donepreviously. The Example attempted to make use of previous work andverify the hypothesis that higher ethanol to acetate feed ratios resultsin higher caprylate production and caprylate specificity. The Examplealso tried to demonstrate that chain elongation and MCC production ispossible using only ethanol as a substrate.

Methods. Bioreactor Configuration, Setup, and Maintenance. The reactorthat was used for the entirety of the experiment was custom designed andconstructed. The reactor is an open culture, anaerobic bioreactor with apertraction unit. The bioreactor itself is an open culture bioreactorthat was originally inoculated with a population from a similar reactorthat was fed corn beer.

Feed and Media. The feed of the bioreactor is composed of a syntheticversion of the effluent expected from a syngas fermenter. The media isprimarily composed of ethanol and acetate when included (as substratesfor reverse beta-oxidation), a yeast extract, and tap water. Additionalchemicals are used in lower concentrations, including chlorides,sulfates, phosphates, and various vitamins. The concentrations ofethanol and acetate are described later, in Experiment Design.

The feed is stored in a refrigeration unit which keeps the feed between2 to 8 degrees Celsius. The influent is fed semi-continuously; it iskept on for 15 minutes and then kept off for 15 minutes. The feed pumpwas set to 1.6 L per day.

Bioreactor. The reactor is kept at both a steady pH of about 5.25 and asteady internal temperature of about 30 degrees Celsius. The pH ismaintained by a pump that adds 0.5 M HCl when the pH falls out of range.Throughout the time of the experiment, the pH was kept at 5.25±0.10. Thereactor is heated by a jacket that pumps heated water down the length ofthe cylindrical reactor. The jacket temperature is kept at about 40°C.±2° C., and the internal temperature of the reactor is kept at about30° C.±1.5° C. For the duration of the following experiment, the reactorwas kept at these temperatures, pH, and pump settings.

The bioreactor itself is filled with a packing material to promotebiofilms. The bioreactors effective volume, taking into account thepacking material, is ˜0.7 L. A unit at the top of the reactor measuresgas production.

Additionally, biomass samples were taken once per week, approximatelytwice per distinct feed ethanol to acetate ratio. Biomass samples weretaken from the middle and bottom of the reactor. This procedure may havedisturbed the ecological community of the bioreactor.

Pertraction Unit. The pertraction unit actively strips hydrophobicmedium chain carboxylic acids from the reactor across a membrane intomineral oil with 30 g/L tri-n-octylphosphine oxide (TOPO). The loadedoil is then pumped to a second membrane pass where an alkali strippingsolution deprotonates the acids into carboxylates, making themhydrophilic in the process. The hydrophilic carboxylates remain in thestripping solution as the unloaded oil returns to the first membrane.The membranes used to load and unload the oil are Liqui-Cel ® MembraneContractors, (product number S08057330; constructed in 2014). Themembranes used to filter out particulates are Pentek GS-6 SED/5, UpperSaddle River, N.J., USA 5 micron sediment and rust particulate filters.The flow rate of the pumps used throughout the pertraction unit are setto 0.81 L/day.

The stripping volume of the reactor is maintained roughly at ˜3.2 L. Thestripping solution is kept above a pH of 9.00 by the use of a pump thatadds 5M NaOH when the stripping pH falls below a pH of 9.00. Thestripping solution is generally changed when the concentration ofcaprylate or another carboxylate is above 0.2M. The procedure for thisevent calls for turning off the stripping, pouring out most of thesolution, leaving 0.45 L of the solution, and diluting that solutionwith DI water. Three times during the experiment the stripping solutionwas changed, on day 180, 201, and 222. For all three times, the newconcentration after equilibrating was taken as zero and subsequentaccumulation of carboxylates was added to the previous total.

Experimental Design. In order to observe the relationship between themolar ratio of ethanol to acetate in the feed to the production ofmedium chain carboxylates in an anaerobic bioreactor, the followingexperiment was designed. Using the bioreactor described in the passageabove, the media fed to the reactor was composed of different ethanol toacetate molar ratios. During the first period of the experiment, thereactor was fed the molar ratio that it was fed for the previous monthsof 10:1 ethanol to acetate. After two weeks, the media would then beswitched to a pure ethanol solution, while maintaining the same organicloading rate measured in g COD/L-day. Subsequently, the reactor feedethanol to acetate molar ratio was decreased incrementally approximatelyevery two weeks to 9:1, then 8:2, then 7:3, then 6:4, and finally 5:5(the lowest possible feed that was previously determined to facilitatereverse beta oxidation). After dropping to a 5 to 5 molar ethanol toacetate ratio, the ratio will be increased for a brief time to 8 to 2molar ratio, followed by a prolonged time of pure ethanol feed toachieve steady state.

The initial feed of the system (10:1) was composed of 300 mM of ethanoland 30 mM of acetate. The corresponding total organic loading rate ofthe system for the entire experiment was calculated to be approximately25 gCOD/L from a hydraulic retention time of about 1.3 days. Theconcentrations of ethanol and acetate in the feeds of the subsequentperiods are listed below (Table 3).

TABLE 3 EtOH:Ace Ethanol Acetate (mol ratio) (mM) (mM) Days Dates (2016)10 to 1  300 30 179-198 Feb 11-March 1 10 to 0  320 0 198-208 March 1-149 to 1 298 33 208-226 March 14-29 8 to 2 274 68.5 226-240 March 29-April12 7 to 3 248.7 106.6 240-257 April 12-29 6 to 4 221.3 147.6 257-275April 29-May 17 5 to 5 191.8 191.8 275- May 17-present

During the course of each period, samples were taken from the feed, thereactor broth, and the stripping solution of the bioreactorapproximately four times a week. The concentrations of ethanol and thecarboxylates were then determined by running the samples through twodifferent GCs (solvents and VFAs respectively). The minimum limits ofdetection for the reactor broth concentrations were 0.05 gCOD/L forethanol and about 0.02 gCOD/L for carboxylates. The minimum detectionlimits in the stripping was 0.02 gCOD/L for the carboxylates.

Over the course of the experiment, twice media was unsuccessfully addedto the system. The first time, the temperature of the refrigeratordropped below freezing and the media froze in the middle of the 7:3period. The media then melted and remained for the duration of the run.The second time was during the addition of the new 6:4 media, when thefeed tubing was bent and feed did not flow to the reactor. The feedtubing was shortened and the media stayed for the remainder of theduration.

Three times during the experiment the stripping solution was changed, onday 180, 201, and 222 (10:1, 10:0, and 9:1 respectively). For all threetimes, the new concentration after equilibrating was taken as zero andsubsequent accumulation of carboxylates was added to the previous total.

Calculations. A number of equations and methods were used to determinedifferent variables:

Hydraulic retention time (HRT): The hydraulic retention time wasdetermined by measuring the mass of effluent over a determined time. Theeffluent density was then approximated to be 1 gram per mL, and thehydraulic retention time was calculated by dividing the volume of thereactor (0.7 L) by the volumetric flow rate (proportional to massdivided by time).

Organic loading rate (OLR): The organic loading rate was determined bydividing the total concentration of the feed (the sum of ethanol,acetate, and yeast in g COD/L) by the hydraulic retention time (HRT).

Transfer Rate of carboxylates: The transfer rate of carboxylates wasdetermined by first multiplying the concentration in the stripping bythe volume of the stripping solution, resulting in gCOD. This data wasgraphed over time for the entire period. A linear regression wasdetermined using Microsoft Excel's LINEST function. The slope (ingCOD/day) was then divided by the reactor volume, giving the transferrate.

Effluent Rate of carboxylates: The effluent rate of the carboxylates wasdetermined by averaging the concentration of the carboxylates ineffluent and then dividing by the hydraulic retention time.

Production Rate of carboxylates: The total production rate is the sum ofthe transfer and the effluent rates for each respective carboxylate.

Conversion Efficiency: The conversion efficiency was found by dividingthe production rate of the carboxylate by the organic loading rate.

Specificity of carboxylates: The specificity of a carboxylate wasdetermined by dividing the production rate of the carboxylate by the sumof all of the production rates.

Pertraction Efficiency: The pertraction efficiency was determined bydividing the transfer rate of a carboxylate by the production rate ofthe carboxylate.

Confidence Intervals. The 95% confidence intervals for the hydraulicretention time, organic loading rate, and effluent concentrations weredetermined by the following equation, where N are the samples:

${{Conf}.\mspace{14mu}{Interval}} = {\frac{{STDEV} \cdot {S(N)}}{\sqrt{{Count}(N)}}*1.96}$

The 95% confidence interval for the transfer rate was determined byfinding the standard error of the slope approximation using Excel'sLINEST function and multiplying it by 1.96 (the t-value of a 95%confidence interval).

The 95% confidence intervals for the effluent rate and conversionefficiency, as they are the product of two variables (one being aninverse), was determined by the following equation.

${{Conf}.\mspace{14mu}{Interval}} = {1.96*\sqrt{a^{2} + \left( \frac{{2*{conf}},b}{3.92} \right)^{2} + {b^{2}*\left( \frac{{2*{conf}},a}{3.92} \right)^{2}}}}$

It is assumed in the equation above that the two variables (a and b) areindependent for simplicity, which is not necessarily true.

For the production rate, being the sum of two variables, the confidenceinterval was determined with the following equation:

${{Conf}.\mspace{14mu}{Interval}} = {1.96*\sqrt{\left( \frac{{2*{conf}},a}{3.92} \right)^{2} + \left( \frac{{2*{conf}},b}{3.92} \right)^{2}}}$

Discussion. The results of the following experiments clearly demonstratea link between the molar ratio of ethanol to acetate in the feed and theproduction of higher carbon density medium chain carboxylates by reversebeta oxidation. As the molar ratio of ethanol to acetate in the feed wasdecreased, the conversion efficiency, specificity, and production rateof caprylate decreased rather dramatically and noticeably. Conversely,the conversion efficiency, specificity of butyrate increased as themolar ethanol to acetate ratio decreased. Being in between butyrate andcaprylate in the reverse beta oxidation chain elongation reaction, itwas predicted that initially the production rate of caproate would go upwhile the production rate of caprylate went down followed by the rate ofcaproate production going down itself. This result can be seen in FIGS.21, 25, 26, and 28.

The maintained high pertraction efficiency for caprylate and the highcaproate pertraction efficiency in the lower ethanol to acetate ratios(FIG. 29 and Table 3) and the either constant or decreasingconcentrations in the effluent (FIGS. 22 and 23) together show that theproduction of these MCCs are most likely not limited by the masstransfer across the membranes into the stripping, but instead arelimited by the production rates of the bacterial communities themselves.

In addition to the increasing caproate production and conversion from afeed ratio of 10:0 to 8:2, the overall production rate of MCCs increasefrom 10:0 to 9:1 to 8:2. This results in the maximum production rate ofcarboxylates (outside of the initial 10:1 period) coming when the feedwas an 8:2 molar ratio of ethanol to acetate. After this point, thetotal production and conversion of carboxylates starts to go down.During this time, caproate production is increasing faster thancaprylate production is decreasing, and after the 8:2 ratio, caproateand caprylate production is decreasing faster than butyrate isincreasing. It should be noted that the pertraction efficiency ofbutyrate is significantly lower than caprylate or caproate for theentirety of the experiment. It is possible that the stripping solutioncannot strip the broth (which the stripping was intended for MCCs), andevidence of this can be seen in FIG. 22 as the broth concentration ofbutyrate continually increases.

Reverse Beta Oxidation and pure Ethanol Feed. The results of theexperiment demonstrate the kinetics of reverse beta oxidation and in thefuture could be analyzed further. The initiation of reverse betaoxidation is limited by the ethanol and acetate substrates. When acetateconcentrations are sufficiently low enough to slow down the initiationof a new carboxylate by reverse beta oxidation, the high ethanolconcentrations of the environment would continue to facilitate thepropagation of reverse beta oxidation by the oxidation of ethanol,extending the carboxylates. As seen in FIG. 20, as the switch to pureethanol decreases the concentration of butyrate and caproate, caprylateconcentrations remain relatively unaffected.

Additionally in the pure ethanol feed ratio, it appears in FIG. 20 asthough the concentration of acetate increases while caproate andbutyrate go down. An explanation is that in the absence of acetate fromtheir environment, the bacteria may have responded by increasing theproduction of acetate before reaching a steady state.

It should be noted further that this experiment not only gives evidencethat a pure ethanol feed could work for a carboxylate bioreactor, but infact that it is effective in giving higher carbon density carboxylates.

Comparisons to Other Studies with 10:1 Molar Ethanol to Acetate Ratios.The results of the period before moving to the pure ethanol feed areinteresting in comparison to previously reported results attained bysimilar feed ratios. The conversion efficiency of caprylate from theresults of the previously reported experiments (68.7%) was in facthigher than the conversion efficiency of caprylate (55.9%) from theconditions that gave the highest reported caprylate production rate atan OLR of 34.7 gCOD/L-day. The conversion efficiency found here thoughis lower than the conversion efficiency (70.7%) which provided thehighest caprylate to caproate production ratio in the previouslyreported (25:1 based on g COD) at an OLR of 15.0 g COD/L-day. The totalconversion efficiency of medium chain carboxylates during thepre-experiment phase (83.5%) was higher than previously reportedproduction of caprylate to caproate ratios (60.8% and 73.3%respectively).

EXAMPLE 3

This example provides a description of methods and systems of thepresent disclosure.

Our bioreactor had been batch-fed semi-continuously through an operatingperiod of 4 years. We used the carboxylate platform to convert the cornbeer fermentation broth to medium chain carboxylic acids (MCCAs). In thefourth year, we performed no experiments, and solely operated thebioreactor to keep the biomass active. More surprising, yet, was thatthe production rate of n-caprylate (C8) was several times higher thanpreviously, and higher than n-caproate (C6) because the pH of bioreactorbroth had jumped from 5.5 to 6.75. Accordingly, we studied the effect ofpH on the product rate of n-caproate and n-caprylate from corn beer(ethanol) fermentation with a 5-L anaerobic sequencing batch reactor(ASBR) with continuous pertraction (membrane-based liquid-liquidextraction).

The operating period consisted of five experimental phases based ondifferent pH levels in the bioreactor (5.5, 6.0, 6.25, 6.75 and 7.0). Westarted with a pH of 6.75, and then decreased the pH to 6.25 and then to5.5. After those phases were completed, we increased the pH again to 6.0and then to 7.0. Each phase was operated for at least 3 hydraulicretention times (HRTs) after the system was found to operate in a stableperformance. During all phases, the temperature of ASBR was controlledat 30±1° C. by circulating heated water through an external heatingjacket of the glass bioreactor. The bioreactor was fed semi-continuously(every 2 days) with corn beer fermentation broth (containing yeast).That was diluted with tap water (diluted 3.4 times). The hydraulicretention time (HRT) was 15 days. The organic loading rate (OLR) ofsystem was 7.66 g COD L⁻¹ d⁻¹ for the phase with a pH of 6.75 and 6.92 gCOD L⁻¹ d⁻¹ for the rest of the 4 phases. The MCCAs were recovered fromthe bioreactor using a pertraction system (liquid-liquid extraction)similar to previous reports.

The production rate of n-caprylate decreased gradually from 1.47±0.05 to1.1±0.01 g L_(bioreactor) ⁻¹ day⁻¹ when the pH of bioreactor decreasedfrom 6.75 to 6.25, and then to 0.73±0.13 g L_(bioreactor) ⁻¹ day⁻¹ at apH of 5.5. When we subsequently increased the pH again from 5.5 to 6.0and then to 7.0, the production rate of caprylate increased again,reaching 1.1±0.15 g L_(bioreactor) ⁻¹ day⁻¹ and 1.49±0.11 gL_(bioreactor) ⁻¹ day⁻¹, respectively (FIG. 31A). This indicated thatbetween a pH of 5.5 to 7.0 that we tested, the highest n-caprylateproduction occurred at the highest pH levels.

In the broth, the concentration of n-caprylate decreased gradually from51.3±2.6 to 2.55±0.54 mM when pH decreased from 6.75 to 5.5. Expectedly,the concentration of n-caprylate increased from 2.55±0.54 mM to 25.5±1.4mM when pH was increased from 5.5 to 7.0 (FIG. 31B). Similarly, theconcentration of total combined MCCAs in the broth was lowest (21.6±1.8mM) during the period of pH 5.5 in bioreactor.

At the end of each phase, we lowered the pH of the stripping solutionwith sulfuric acid, which spontaneously separates the MCCA oil from thestripping solution through phase separation. The main components of theMCCA oil were n-caproic acid and n-caprylic acid (other fatty acids werenot detected, ≤GC detection limit: 0.12 mM). The results showed that themolar percentage of n-caprylate gradually increased from 75% to 97% whenpH of bioreactor increased from 5.5 to 7.0 (FIG. 32).

In summary, the highest relative n-caprylate production occurred at thehighest pH level of 7.0, while the highest relative n-caproateproduction occurred at the lowest pH of 5.5 (less green in FIG. 31).This can be explained by the differences for both the toxicity and theextraction rates between undissociated n-caprylic acid of n-caprylicacid at different pH levels (and their interactions). Since the pKa ofn-caproate and n-caprylate is around 4.9 (and possibly a bit higher dueto micel formation in the bioreactor, especially for n-caprylate), therelative level of undissociated acids is higher at the lower pH levelsof the Example. Undissociated n-caproic acid and n-caprylic acid arehighly toxic to the community, especially for n-caprylic acid that istoxic at an approximately 10× lower concentration than n-caproic acid,while the dissociated salt is not toxic at all. That is why we operatein-line extraction systems for our bioreactors to keep theconcentrations in the bioreactor (and thus effluent) to a minimum.Therefore, at the lower pH levels in this Example n-caproate would notbe further converted to n-caprylate as much due to high toxicityproblems for n-caprylic acid than at the higher pH levels.

At the same time, the extraction rates for undissociated n-caprylic acidare much higher than for undissociated n-caproic acid due to the muchmore oily nature of n-caprylic acid compared to n-caproic acid (themaximum solubility concentration is about 10× lower for n-caprylic acidcompared to n-caproic acid). Pertraction of n-caproic acid has to occurat the lower pH levels. We found here that n-caprylic acid can beextracted at a pH of 7. It is possible that the observed pKa is higherin our bioreactor for n-caprylic acid due to micel formation, but thisdoes need to be investigated further. Therefore, only the longerchemical (C8) n-caprylic acid can be extracted at a pH of 7.0 (97% inFIG. 32) and automatically the system selects for this n-caprylic acidproduction at the higher pH levels. Thus, both the lower toxicity ofn-caprylic acid and the higher extraction of n-caprylic acid compared ton-caproic acid at the higher pH levels explain why the relativeproduction of n-caprylate is preferred at a neutral pH compared to amidly-acidic pH.

We had operated the bioreactors at a mildly acidic pH level of 5.5 (oreven lower) in the past to prevent acetoclastic methanogens to convertall substrate and ethanol into methane. Thus, at a pH of 7.0 we wouldhave anticipated a methanogenic anaerobic digester rather than a chainelongating bioreactor. Through separate experimentation with small serumbottles we found that the concentrations of undissociated n-caprylicacid were toxic to these methanogens. This explains why we can chainelongate at the higher pH levels without adding amethanogenic-inhibiting compound such as bromo-ethanesulfonate (BrES).We added anaerobic digester biomass from an active digester to the pH 7bioreactor and we did not observe a shift to a methanogenic system evenafter waiting one month (no change). This substantiates the claim thatwe can operate a chain elongating bioreactor sustainably at a pH levelof 7.0.

EXAMPLE 4

This example provides a description of methods and systems of thepresent disclosure.

In this example, we examined the effect of different ethanol-to-acetatesubstrate molar ratios on the production of medium chain carboxylates.Higher ethanol-to-acetate substrate molar ratios led to higherselectivity for n-caprylate. The highest n-caprylate selectivity in thisExample occurred when the substrate contained primarily ethanol(ethanol-to-acetate molar ratio >100), however, the overall medium chainproductivity of the bioreactor declined. At an approximately one to onesubstrate molar ratio, n-caprylate production stopped in the bioreactor.Finally, Illumina 16S rRNA gene sequencing of the bioreactor microbiomeacross time and in two sampling locations on the bioreactor revealed arelatively uneven microbiome that was dominated by Firmicutes andProteobacteria phyla members.

Here, our main objective was to experimentally determine what substrateethanol-to-acetate ratio was optimal to promote n-caprylate productionin a continuously operated bioreactor with product extraction. Inaddition, this Example aimed to investigate whether n-caprylate could beproduced from primarily ethanol in the substrate (i.e.,ethanol-to-acetate substrate molar ratio >100). A previously publishedthermodynamic model was extended in this example to n-caprylate and theexperimental data collected was used to validate the model. Finally,this example sed Illumina 16S rRNA gene sequencing to investigate whatOTUs were present in the bioreactor microbiome during the operatingperiod and correlated the relative abundance of these OTUs withn-caprylate specificities.

MATERIALS AND METHODS. Bioreactor Setup. The bioreactor system that wasoperated in this example was also used in other examples and isdescribed herein. Briefly, an upflow anaerobic filter (working volume0.7 L) was operated with a continuous in-line, membrane-basedliquid-liquid extraction (i.e., pertraction) system. The feed rate usedin this Example was approximately 0.6 L d⁻¹, while the system recycleflow rate was 130 L d⁻¹, which resulted in a recycle feed ratio of over200. The pH of the bioreactor broth was maintained at 5.26±0.09 viaaddition of 0.5 M hydrochloric acid to the well-mixed feed and recycleinlet at the base of the bioreactor. The temperature of the bioreactorwas maintained at 30±1° C. The hydraulic retention time used in thisExample was ˜1.2 days. The pH of the alkaline extraction solution wasmaintained at 9.48±0.34 via addition of 5 M sodium hydroxide.

Growth Medium and Inoculum. The growth medium used in this Example isknown in the art and has been described previously. For each operatingperiod, the substrate concentrations of ethanol and acetate were variedto achieve the targeted substrate molar ratios of ethanol and acetate,while maintaining organic loading rates in the range of 18.7 to 28.2 gCOD L⁻¹ d⁻¹ (Table 4). As mentioned above, this bioreactor microbiomewas also used in another example described herein. No new inoculum wasadded to the bioreactor between studies.

TABLE 4 Operating data for the bioreactor. Average hydraulic retentiontime (HRT), influent ethanol and acetate concentration, substrate molarratios (ethanol-to-acetate), and organic loading rates (OLR) per periodare reported as mean ± s.e. Substrate Molar Ethanol Acetate OLR PeriodDays HRT Ratio (mM) (mM) (g COD L⁻¹ d⁻¹) Period 1 155 to 198  1.2 ± 0.02 7.8 ± 0.65 200.42 ± 9.42 25.68 ± 1.75 18.72 ± 0.81 Period 2 198 to 2111.17 ± 0.05 183.29 ± 38.86  289.14 ± 3.64  1.58 ± 0.33 25.15 ± 1.1 Period 3 211 to 227 1.27 ± 0.07 11.29 ± 1.38  281.97 ± 5.21 24.97 ± 3.0123.88 ± 1.43 Period 4 227 to 240 1.16 ± 0.04 4.45 ± 0.29 281.69 ± 10.563.35 ± 3.35 28.17 ± 1.24 Period 5 240 to 257 1.17 ± 0.04 2.43 ± 0.17 228.81 ± 10.65 94.09 ± 4.74 25.19 ± 1.18 Period 6 257 to 275  1.2 ±0.05 1.93 ± 0.08 216.13 ± 5.26 111.83 ± 3.41  24.63 ± 1.08 Period 7 275to 291  1.2 ± 0.04 1.22 ± 0.06 177.87 ± 4.53 146.25 ± 6.24  23.34 ± 0.85

Bioreactor Operation. In the main phase of this example, we operated thebioreactor at the following substrate (ethanol-to-acetate) molar ratios:7.8, 137.6, 11.3, 4.5, 2.5, 1.9, and 1.2 (Table 4). We operated thebioreactor at each ratio for a period of at least two weeks (at least 11HRTs). Following the loss of performance at the 1.2 molar ratio, we rananother set of similar substrate molar ratios: 3.4, 4.4, and 90.1. TheHRTs and OLRs for these later molar ratios, which are not the main focusof the paper, can be found in Table 5.

Liquid and Gas Analysis. Liquid samples (1.5 mL) were collected from thebioreactor influent, the bioreactor broth, and the alkaline extractionsolution, as has been described in Example 1. Analysis of the samples todetermine carboxylate and ethanol concentrations was carried out by gaschromatography using previously reported methods. The concentrations ofmethane, carbon dioxide, and hydrogen gases (detection limit 0.2%) weremeasured using a previously known GC method/system.

Calculations and Statistical Analysis of Operational Data. Carboxylateproduction rates are calculated as the average values for each operatingperiod. Average effluent production rates (g COD L⁻¹ d⁻¹) and averagetransfer rates via product extraction (g COD L⁻¹ d⁻¹) were summed toyield the total production rates (g COD L⁻¹ d⁻¹). COD stands forchemical oxygen demand. Standard errors were reported. The averageeffluent production rates were calculated by dividing the averagecarboxylate concentration per period by the average HRT for that period.The average HRT per period was calculated based on the average influentflow rate per period, which was determined volumetrically. The averagetransfer rates were calculated by first plotting the increasingconcentrations of the individual carboxylates in the alkaline extractionsolution vs. time. Then the linear model function, 1 m, in R was used todetermine the slope and standard error of the best-fit line throughthese points. The slope was then divided by the bioreactor workingvolume (0.7 L) to get the average transfer rate per period. Wecalculated the conversion efficiency as the individual carboxylate totalproduction rate divided by the organic loading rate per period. Inaddition, specificity was calculated as the individual carboxylate totalproduction rate divided by the combined total production rate for allcarboxylates during each period (where the carboxylates included aren-butyrate, n-caproate, and n-caprylate). Furthermore, pertractionefficiency was calculated as the average transfer rate divided by thetotal production rate for each carboxylate. Finally, RStudio (version1.0.136) was used for all data analyses.

Thermodynamic Model Development. Here, we extended a previouslypublished generalized stoichiometric model to predict the thermodynamicfavorability of n-caprylate formation at different substrate molarratios. FIG. 33 shows the extended model. Details on the modeldevelopment can be found herein under the section entitled“Thermodynamic Model Development”. Briefly, the model usesstoichiometric relationships to predict the moles of caprylate and theamount of ATP that would be produced based on the moles of ethanol andacetate provided to the system. For the purpose of this model, the molesof n-butyrate and n-caproate formed were also fixed, to variables “b”and “c”, respectively. The boundary for the metabolic flux was set to 10moles of ethanol and acetate combined (therefore by setting moles ofethanol is equal to “a” in the model, moles of acetate is set to“10-a”). Based on this stoichiometry and the ethanol and carboxylateconcentrations measured in the bioreactor, the Gibbs free energy of thereaction is calculated as well as the Gibbs free energy required for ATPformation. If the Gibbs free energy of the reaction was more negativethan the Gibbs free energy required for ATP formation, the reaction wasdeemed feasible.

Microbial Community Analysis. We collected biomass samples for Illumina16S rRNA gene sequencing analysis from the bottom and middle of thebioreactor approximately weekly throughout the operating period. Themethod of collecting biomass samples from the bioreactor broth has beenpreviously described with the exception that, in the previous studybiomass samples were only collected from the middle of the bioreactor,whereas in this Example we also collected them from a bottom samplingport. Pelleted biomass samples were stored at −80° C. until furtherprocessing. Genomic DNA was extracted using the PowerSoil-htp 96 WellSoil DNA Isolation kit (MO BIO Laboratories Inc., Carlsbad, Calif.)according to the protocol of the manufacturer. The DNA amplificationprotocol was described previously with the following exceptions:Mag-Bind RxnPure Plus magnetic beads solution (Omega Biotek, Norcross,Ga., USA) was used instead of Mag-Bind E-Z Pure and 50 ng DNA per samplewas pooled instead of 100 ng. It should be noted that duplicate PCRreactions of each DNA extract were performed and pooled prior tosequencing. Paired-end reads were joined in QIIME version 1.9.1 usingthe joined_paired_ends.py script and then the joined reads were uploadedto QIITA (qiita.microbio.me) for further processing. The sortmernamethod was used to bin sequences in operational taxonomic units (OTUs)at 97% identity. Taxonomy was assigned for representative sequencesselected for each OTU using the Greengenes v13.8 database from August2013. The remaining analyses were performed locally in QIIME v1.9.1 andRStudio v1.0.136. Singletons were removed from the dataset resulting in932 unique OTUs.

Alpha diversity was analyzed via Gini coefficient, observed OTUs, andShannon diversity. In addition, heat maps were created to represent OTUrelative abundance via the gplots package in R. Correlations of OTUrelative abundance with n-caprylate specificities was investigated usingthe Spearman's rank correlation coefficient via the Hmisc package inRStudio. Correlations with p>0.001 were considered significant. OnlyOTUs that reached at least 1% relative abundance in any one bioreactorsample were considered in the correlation analysis.

Sequences were submitted to EBI under the following accession number.Sequences and study metadata are publically available in QIITA, which isan open-source microbiome storage and analysis resource.

RESULTS AND DISCUSSION. Higher Ethanol-to-Acetate Molar Ratio inSubstrate Leads to Higher N-Caprylate Specificity. We observed thathigher ethanol-to-acetate molar ratio in the substrate led to highern-caprylate specificity in the products of the bioreactor (FIG. 34,Table 6). Here n-caprylate specificity is defined as the production rateof n-caprylate vs. the combined production rates of n-butyrate,n-caproate, and n-caprylate on a COD basis. The highest n-caprylatespecificity achieved in this example was 82±10% at the highest substratemolar ratio tested, 183.3±38.9 (Table 6). To our knowledge, this is thehighest substrate molar ratio that has been tested in the literature formedium chain carboxylate production. At the lowest substrate molar ratioused in this Example (i.e., 1.2±0.1 moles of ethanol per moles ofacetate), n-butyrate production and specificity was much higher than atthe higher substrate molar ratios tested, while no n-caprylateproduction was observed (FIG. 34, Table 6).

With the exception of Period 1, we decreased the substrate molar ratioduring the operating period from 183.2 in Period 2 to 1.22 in Period 7.When the substrate molar ratio of ethanol to acetate decreased, acetateand n-butyrate concentrations increased in the effluent of thebioreactor (FIG. 35 and Table 7). In Period 2, at the highest substratemolar ratio (183.3±38.9) used in this example, the average acetate andn-butyrate concentrations measured in the effluent leaving thebioreactor were 4.1±0.6 mM and 3.6±0.9 mM, respectively (FIG. 35, Table7). As the substrate molar ratio was decreased throughout the course ofthe Example (Periods 2 to 7), the acetate and n-butyrate concentrationsin the bioreactor increased. At the lowest substrate molar ratioemployed in this Example (1.2±0.6) in Period 7, the average acetate andn-butyrate concentrations in the effluent were 88.2±8.4 mM and 18.3±0.9mM, respectively (FIG. 35, Table 7). Since the bioreactor had a highrecycle ratio of ˜220 (broth recycle flow rate divided by effluent flowrate), the concentration leaving in the effluent can be consideredroughly equivalent to the concentration in the bioreactor.

We extended a previously reported hermodynamic model to predict thethermodynamic favorability of n-caprylate formation at the differentethanol-to-acetate ratios experienced by the bioreactor microbiome.Since the bioreactor was well-mixed, we used the average measuredeffluent concentrations (FIG. 35, Table 7) in this model to representthe closest approximation of the conditions the microbiome saw. Similarto a previously observed trend for n-caproate, our model predicted thathigher ethanol-to-acetate ratios experienced by the microbiome generallylead to more favorable thermodynamic conditions for chain elongation ton-caprylate (FIG. 38). In other words, the Gibbs free energy of then-caprylate formation reaction was more negative than the Gibbs freeenergy required for ATP production at the substrate molar ratios of183.3, 11.3, and 4.4 (which resulted in ethanol-to-acetate ratiosmeasured in the bioreactor of 16.4, 7.0, and 3.1, which we used in themodel), indicating that the formation of n-caprylate wasthermodynamically feasible at these ratios. For the most part, our modeldescribed what we observed experimentally, with the exception of thefirst period of the Example. In Period 1, when the substrate molar ratiowas 7.8±0.7, the n-caprylate conversion efficiency was the highestachieved in our Example (68±7%) (Table 8) and the ethanol concentrationleaving in the effluent was low at 15.6±1.0 mM (Table 7). The relativelyfaster conversion rate of ethanol in this period resulted in arelatively lower ethanol-to-acetate molar ratio measured in the effluentof 3.0 (Table 7). At this ethanol-to-acetate molar ratio and theobserved concentrations of n-butyrate, n-caproate, and n-caprylate, ourmodel predicted that n-caprylate formation becomes thermodynamicallyfeasible (FIG. 38), even though we observed the highest n-caprylateproduction rates in this period (Table 9). Our model is an oversimplification of what occurred in the bioreactor and does not representwhat the microbes see. In addition, our thermodynamic model does notaccount for the kinetics in the bioreactor system. The model also doesnot account for effect of the continuously operating pertraction system,which was continuously and preferentially removing longer chaincarboxylates from the bioreactor environment.

N-Caprylate Production with Primarily Ethanol in Substrate but LowerOverall Productivity. The highest substrate molar ratio resulted in thehighest n-caprylate specificity (FIG. 34), as well as the highestn-caprylate to n-caproate productivity ratio of 5.9±0.7 (Table 6).However, it did not result in the highest conversion efficiency (Table8). In fact, in this period (Period 2), the n-caprylate conversionefficiency (43±5%) was lower than in Period 1 under the 7.8 substratemolar ratio, where the n-caprylate conversion efficiency was 68±7%(Table 8). During Period 2, the ethanol concentration increased in theeffluent of the bioreactor (FIG. 35B). The concentration remained wellbelow the concentrations that have previously been found to beinhibitory. However, it is clear that excess ethanol was not being fullyutilized by the bioreactor. Similar to the results in this Example, itwas also found in Example 1 that a lower n-caprylate productivity in theperiod where the highest n-caprylate to n-caproate productivity ratiowas observed. Thus, it appears that at higher ethanol-to-acetate molarratios there is a trade-off between improved n-caprylate specificity anddecreased overall productivity. In this Example, relatively short HRTs(i.e., 1.2 days) were employed. A longer HRT may allow for improvedproduction at these higher ethanol-to-acetate substrate ratios, becauseless product is washed out.

n-Caproate and n-caprylate contain six or eight carbon atoms in theirchain, respectively. Thus they are relatively hydrophobic and easy toextract from solution, as compared to n-butyrate. Indeed, it can be seenthat the average extraction efficiency per period for n-butyrate rangedfrom a minimum of 11.8% to a maximum of 66.8%, whereas the averageextraction efficiency for n-caproate per period was always greater than65% and for n-caprylate was always greater than 96% in the periods wheren-caprylate production was detected (Table 10). Due to the highextraction efficiency for n-caprylate that we observed in this Example,it is unlikely that are n-caprylate production rates were limited bymass transfer limitations.

Microbiome Shifts Correlated to N-Caprylate Specificity. We performed atime-series analysis of the microbiome that was sampled from twolocations in our bioreactor: a bottom and a middle sampling port. Weused Illumina 16S rRNA gene sequencing to analyze the samples. Similarto the microbial community stratification that has previously beenobserved in upflow anaerobic sludge blanket reactors, we observed cleardifferences in the compositions of the microbiomes sampled from thebottom and the middle of the bioreactor. The majority of samples fromthe bottom of the bioreactor had a higher relative abundance of thephylum Firmicutes compared to the phylum Proteobacteria, whereas thereverse was true in the middle of the bioreactor (FIG. 39). In addition,the bottom of the bioreactor had a more diverse microbiome as indicatedby the Shannon diversity index and Gini coefficient (Table 11). Thoughthe bioreactor was well-mixed due to the high recycle rate employed, itis unlikely that the ethanol and carboxylate concentrations were uniformthroughout the bioreactor. Substrate and acid (0.5M HCl) for pH controlwere added at the base of the bioreactor, which may have caused slightlyhigher concentrations of un-dissociated carboxylic acids and ethanol tobe seen by the microbes at the bottom of the bioreactor, as compared tothe middle. Nevertheless, we did observe common OTUs between the middleand the bottom bioreactor samples, which are indicated on the heat maps(FIGS. 36 and 37). Of the OTUs that reached over one percent relativeabundance in the bioreactor samples from the bottom and middle of thebioreactor (40 and 45 OTUs, respectively) and are shown in the heatmaps, 28 of these OTUs were shared between the two sampling locations.

For both the bottom and the middle of the bioreactor, we examined whichOTUs were positively or negatively correlated with n-caprylatespecificities based on Spearmans rank coefficient (p<0.001) (FIGS. 36and 37). Some common patterns emerged between the two samplinglocations. Two different OTUs belonging to the family Ruminococcaceaewere positively correlated to n-caprylate specificities in one of thetwo locations (OTU ID 720944 in the middle; OTU ID 300620 in thebottom). In addition, a Veillonellaceae family OTU (ID 225954) and anOscillospira OTU (ID 4386437), which belongs to the Ruminococcaceaefamily, were positively correlated to n-caprylate specificities in bothlocations. Members of the Ruminococcaceae family, specifically theClostridium cluster IV, including Oscillospira, have been found to beassociated with: 1) n-butyrate production in the human gut, 2)n-caproate production from lactate in Chinese strong liquorfermentation, and 3) n-caproate, n-caprylate, and trace amounts ofn-decanoate production from biorefinery thin stillage. Another OTU thatis in the Clostridium cluster IV, an OTU belonging to the Anaerofilumgenus (ID 130679), was also positively correlated to n-caprylatespecificities in our bioreactor, though it was only seen in the bottombioreactor samples. In Example 1, using the same bioreactor, members ofthe Ruminococceae family (the same OTUs as ones mentioned above thatwere positively correlated in this Example—IDs 720944 and 300620), aswell as an OTU classified to Oscillospira genus level were also found tobe positively correlated with n-caprylate productivity (the OscillospiraOTU (ID 115035) found in Example 1 was also found in middle and bottombioreactor samples but was not found to be correlated to n-caprylatespecificity). In Example 1, biomass samples were only collected from themiddle of the bioreactor.

Common patterns were also observed between the bottom and middle of thebioreactor in terms of which OTUs were negatively correlated withn-caprylate specificities. Different OTUs classified as Acetobacter spp.(OTU ID 4333237 in the bottom and OTU ID 635373 in the middle), aDesulfosporosinus meridiei OTU (ID 3406110), a Lactobacillus zeae OTU(ID 73609), and an OTU in the family Xanthomonadaceae (ID 588916) werenegatively correlated with n-caprylate specificities. In the middle ofthe bioreactor, a Methanobacterium OTU (ID 2508129) was found to benegatively correlated to n-caprylate specificities. Acetobacter is anobligate aerobe that can convert ethanol to acetic acid. In was observedthat Acetobacter in survived in the anaerobic bioreactors and it ispossible that their survival could be due to trace amounts of oxygenentering in the non-anaerobic feed because we did not attempt to makethe feed line completely anaerobic. It was observed that the populationsof Lactobacillus and Acetobacter spp. declined and the overall mediumchain carboxylate productivity of the bioreactor increased. Thisobservation may be due to the substrates used (ethanol, acetate, andbasal media).

Bioreactor System Unable to Recover Performance After Low SubstrateRatio. Following the lowest substrate ratio 1.2 moles ethanol to molesacetate, the bioreactor never recovered to its prior level ofperformance. In Period 8 (following low ratio), the substrate molarratio was increased to 3.38. Despite the increase in the substrate molarratio, the n-caprylate productivity did not recover to previous levels(Table 9). Medium chain conversion efficiency for this period was only19.6±2.1% (Table 8). In this period, gas production began to increase.Across the main periods of the Example (Periods 1 to 7) the average gasproduction was 0.38±0.01 mL d⁻¹. In Period 8, the gas productionincreased to 1.22±0.06 mL d⁻¹, and by Period 11 the gas productionincreased to 2.74±0.04 mL d⁻¹ (Table 12). It is possible that at the lowsubstrate molar ratio in Period 7 (1.2), methanogens were able to takeover the system and shift the bioreactor away from medium chaincarboxylic acid production.

The microbiome in this bioreactor is shaped to efficiently producemedium chain carboxylates by lowering the pH and using productextraction. In a previous study, which used a similar setup to producemedium chain carboxylates from corn beer, it was observed that highern-caproate productivity correlated with a more uneven microbiome.Similarly the community in our bioreactor was relatively uneven (Table11). Shaping the bioreactor to produce medium chain carboxylates at highrates and efficiencies may come at the price of decreased bioreactorstability, since it took so long to regain stability. Work is needed tofurther study these observations, and to find operating conditions thatcan mitigate this problem of this non-resistant community.

Conclusions. In conclusion, this Example demonstrated that higherethanol-to-acetate substrate molar ratios lead to higher n-caprylatespecificities. At very low ethanol-to-acetate substrate molar ratio(˜1), no n-caprylate production was observed. To our knowledge, this isthe first Example to demonstrate efficient n-caprylate production fromprimarily ethanol in the substrate (i.e., ethanol-to-acetate molar ratiogreater than 100), though it should be noted that the n-caprylateconversion efficiency was lower at this ratio than at lower ratiostested. This Example also characterized the microbiome present at thedifferent substrate molar ratios tested and examined which microbes werecorrelated with improved n-caprylate specificity in the bioreactor.

SUPPLEMENTARY METHODS. Thermodynamic Model Development. As mentionedherein, the previously published generalized stoichiometric modelpredicted the thermodynamic favorability of n-caproate formation atdifferent ethanol-to-acetate substrate molar ratios. In this Example, weextended the model to predict n-caprylate formation at differentethanol-to-acetate substrate molar ratios. The model uses stoichiometricrelationships to predict the moles of n-caprylate and the amount of ATPthat would be produced based on the moles of ethanol and acetateprovided to the system. The boundary for the metabolic flux in the modelwas set to 10 moles of carbon moieties consumed. In our model, moles ofethanol is set to “a” and therefore, the moles of acetate is 10-a. Forthe purpose of this model, the moles of n-butyrate and n-caproate formedwere also fixed, as variables “b” and “c”. In our model, thestoichiometry of all metabolites (i.e., ethanol, acetate, n-butyrate,n-caproate, and n-caprylate, molecular hydrogen, water, intermediarymetabolites, redox mediators, and ATP) depends on the variables “a”,“b”, and “c”. The stoichiometry for re-oxidation of reduced ferredoxinvia H2-ase or Rnf and ATP synthase varies depending on these variables,which determines the molecular hydrogen production and ATP production,respectively. Based on this stoichiometry, as well as the ethanol andcarboxylate concentrations measured in the bioreactor, the Gibbs freeenergy of the reaction is calculated as well as the Gibbs free energyrequired for ATP formation. If the Gibbs free energy of the reaction wasmore negative than the Gibbs free energy required for ATP formation, thereaction was deemed feasible. For the purpose of these calculations thestandard Gibbs free energies were taken from Kleerebezem and vanLoosdrecht.

TABLE 5 Operating data for later periods for the bioreactor. Averagehydraulic retention time (HRT), influent ethanol and acetateconcentration, substrate molar ratios (ethanol-to- acetate), and organicloading rates (OLR) per period are reported as mean ± s.e. SubstrateMolar Ethanol Acetate OLR Period Days HRT Ratio mM (mM) (g COD L⁻¹ d⁻¹)Period 8 291 to 337 1.14 ± 0.04 3.38 ± 0.35 218.15 ± 9.68  64.64 ± 6.1 23.46 ± 1.16 Period 9 337 to 358  1.2 ± 0.04 4.35 ± 0.35 245.15 ± 11.9256.39 ± 3.55   24 ± 1.31 Period 10 358 to 370 1.14 ± 0.04 4 220.82 ±17.14 NA 24 Period 11 370 to 397 1.12 ± 0.05 90.14 ± 12.08 286.51 ±6.08   3.18 ± 0.42 26.22 ± 1.35

TABLE 6 Specificities per period (n-butyrate, n-caproate, or n-caprylateProduction vs. total production of these carboxylates) and C8 to C6ratio Substrate Specificity (%) Period Molar Ratio C8 to C6 RatioN-Butyrate N-Caproate N-Caprylate Period 1  7.8 ± 0.65 3.04 + 0.323.57 + 0.4  23.85 + 2.05 72.57 + 8.2  Period 2 183.29 ± 38.86  5.92 +0.74 4.21 + 0.98 13.85 + 1.57 81.94 + 10.17 Period 3 11.29 ± 1.38 1.95 + 0.42  3.7 + 0.91 32.64 + 5.45 63.66 + 14.03 Period 4 4.45 ± 0.290.67 + 0.07 14.2 + 1.54 51.34 + 3.33 34.47 + 3.61  Period 5 2.43 ± 0.170.63 + 0.16 33.38 + 3.81  40.97 + 6.41 25.65 + 5.96  Period 6 1.93 ±0.08 0.66 + 0.13 40.51 + 3.89  35.8 + 4.5 23.69 + 4.31  Period 7 1.22 ±0.06 NA 97.18 1.67 NA Period 8 3.38 ± 0.35 1.23 + 0.23 64.44 + 3.71 15.93 + 1.75 19.62 + 3.12  Period 9 4.35 ± 0.35 0.13 + 0.01 92.97 +5.72   6.25 + 0.47 0.78 + 0.07 Period 4   1 + 0.31 59.24 + 12.78 20.34 +2.51 20.42 + 6.86  10 Period 90.14 ± 12.08 0.46 + 0.22 56.09 + 7.56 29.99 + 6.41 13.92 + 6.14  11

TABLE 7 Average effluent concentrations (mM) in bioreactor per period(mean ± s.e.). nd if concentration was below detection limit (0.2 mM).If only one measurement was above detection limit, only that measurementis reported. Valerate, isocaproate, and heptanoate were not detected inthe bioreactor during the Example. Concentration in Effluent (mM) (mean± s.e.) Period Ethanol Acetate Propionate Isobutyrate N-ButyrateIsovalerate N-Caproate N-Caprylate P1 15.6 ± 1   5.1 ± 0.3 nd 0.4 ± 04.7 ± 0.4 nd 4.5 ± 0.2 1.5 ± 0.1 P2 67.3 ± 8.8  4.1 ± 0.6 nd 0.4 ± 0 3.6± 0.9 0.4 2.9 ± 0.4 1.4 ± 0.1 P3 88.3 ± 1.9 12.6 ± 1.5 nd nd 2.5 ± 0.2nd 2.8 ± 0.2 0.9 ± 0.1 P4 49.7 ± 4.8  16 ± 1.5 nd 0.4 ± 0  9 ± 1.3 nd2.5 ± 0.2 0.3 ± 0.1 P5 48.5 ± 4.3 35.6 ± 2.2 nd 0.5 ± 0 18.1 ± 0.6  nd0.9 ± 0.1 nd P6 54.8 ± 4  56.3 ± 4.9 nd nd  20 ± 1.2 nd 0.5 ± 0  nd P749.7 ± 3.2 88.2 ± 8.4 nd 0.4 18.3 ± 0.9  nd 0.4 ± 0.0 0.2 P8 58.5 ± 5.328.6 ± 5.4 0.5 ± 0 0.7 ± 0  28 ± 1.4 nd 0.5 ± 0  0.2

TABLE 8 Conversion efficiencies (individual carboxylate production ratedivided by organic loading rate) for n-butyrate, n-caproate,n-caprylate, and the combined medium chain carboxylic acids (MCCA;includes n-caproate and n-caprylate) per period. Substrate ConversionEfficiency (%) Period Molar Ratio N-Butyrate N-Caproate N-Caprylate MCCAP1  7.8 ± 0.65  3.36 ± 0.33 22.46 ± 1.55 68.34 ± 6.87  90.8 ± 7.44 P2183.29 ± 38.86   2.21 ± 0.49  7.29 ± 0.67 43.11 ± 4.52  50.4 ± 4.7 P311.29 ± 1.38   2.24 ± 0.5 19.75 ± 2.53 38.53 ± 7.42 58.28 ± 8.19 P4 4.45± 0.29  9.75 ± 1.06 35.25 ± 2.3 23.67 ± 2.49 58.92 ± 3.84 P5 2.43 ± 0.1723.36 ± 2.15 28.67 ± 4.05 17.95 ± 3.99 46.62 ± 5.88 P6 1.93 ± 0.08 32.65± 2.76 28.85 ± 3.38 19.09 ± 3.36 47.95 ± 4.99 P7 1.22 ± 0.06 21.38 ±3.52  0.37 ± 0.02  0.25 ± NA  0.62 ± NA P8 3.38 ± 0.35  35.5 ± 2.23 8.78 ± 0.99 10.81 ± 1.74 19.59 ± 2.11 P9 4.35 ± 0.35 33.24 ± 2.35  2.23± 0.19  0.28 ± 0.03  2.51 ± 0.2 P10 4  47.6 ± NaN 16.34 ± NaN 16.41 ±NaN 32.74 ± NaN P11 90.14 ± 12.08 18.53 ± 1.97  9.91 ± 1.95  4.6 ± 1.9914.51 ± 2.83

TABLE 9 Production rates of n-butyrate, n-caproate, and n-caprylate.Total production rate is sum of effluent and transfer rate. C8 to C6production ratio is also reported. In Period 7 transfer rate for bothn-caproate and n-caprylate was negative based on stripping data so setto zero. N-Caprylate effluent measurement only had one value. Thereforecould not calculate C8 to C6 ratio. In P9 stripping was off. N-ButyrateProduction N-Caproate Production N-Caprylate Production (g COD/L/d) gCOD/L/d) Rate (g COD/L/d) Effluent Transfer Total Effluent TransferTotal Effluent Transfer Total Period Rate Rate Rate Rate Rate Rate RateRate Rate P1 0.63 ± 0.06 NA 0.63 ± 0.06 0.96 ± 0.04 3.25 ± 0.22 4.21 ±0.23 0.44 ± 0.02 12.36 ± 1.16  12.8 ± 1.16 P2 0.49 ± 0.12 0.07 ± 0.030.56 ± 0.12 0.64 ± 0.1  1.19 ± 0.11 1.83 ± 0.15 0.41 ± 0.02 10.43 ±1.03  10.84 ± 1.03  P3 0.31 ± 0.03 0.22 ± 0.11 0.53 ± 0.11 0.57 ± 0.054.15 ± 0.53 4.72 ± 0.54 0.25 ± 0.03 8.95 ± 1.69  9.2 ± 1.69 P4 1.24 ±0.18 1.51 ± 0.21 2.75 ± 0.27 0.55 ± 0.05 9.38 ± 0.48 9.93 ± 0.48  0.1 ±0.04 6.57 ± 0.64 6.67 ± 0.64 P5 2.46 ± 0.11 3.42 ± 0.45 5.88 ± 0.47  0.2± 0.02 7.03 ± 0.96 7.22 ± 0.96 NA 4.52 ± 0.98 4.52 ± 0.98 P6 2.67 ± 0.195.37 ± 0.55 8.04 ± 0.58 0.11 ± 0.01   7 ± 0.77 7.11 ± 0.77 NA 4.7 ± 0.84.7 ± 0.8 P7 2.44 ± 0.14 2.55 ± 0.79 4.99 ± 0.8  0.09 ± 0   0 0.09 ± 0  0.06 0 0.06 P8 3.94 ± 0.24 4.39 ± 0.22 8.33 ± 0.32 0.12 ± 0.01 1.94 ±0.21 2.06 ± 0.21 0.06 ± NA 2.47 ± 0.39 2.54 ± 0.39 P9 7.98 ± 0.36 NA7.98 ± 0.36 0.54 ± 0.03 NA 0.54 ± 0.03 0.07 ± 0.01 NA 0.07 ± 0.01 P104.38 ± 0.36 7.04 ± 1.99 11.42 ± 2.02  0.06 ± 0   3.86 ± 0.04 3.92 ± 0.04NA 3.94 ± 1.23 3.94 ± 1.23 P11 1.23 ± 0.08 3.63 ± 0.44 4.86 ± 0.45 0.14± 0.04 2.46 ± 0.49  2.6 ± 0.49 0.08 ± 0 1.13 ± 0.52 1.21 ± 0.52

TABLE 10 Extraction efficiencies per period (mean and s.e.) Extractionsystem was off for P9. Average Extraction Efficiency (%) PeriodN-Butyrate N-Caproate N-Caprylate P1 NA 77.25 + 6.71  96.6 + 12.63 P211.75 + 5.61 65.18 + 7.93 96.23 + 13.23 P3  41.6 + 22.62  87.98 + 15.0797.27 + 25.55 P4 54.86 + 9.25 94.46 + 6.6   98.5 + 13.38 P5 58.13 + 8.97 97.28 + 18.56   100 + 30.72 P6 66.77 + 8.36  98.49 + 15.23   100 +24.12 P7  51.02 + 17.84 NA NA P8 52.67 + 3.38  94.24 + 13.91 97.53 +21.39 P9 strip off strip off strip off P10  61.65 + 20.59 98.53 + 1.46  100 + 44.19 P11  74.63 + 11.48  94.73 + 26.15 93.71 + 58.95

TABLE 11 Alpha Diversity Metrics; bottom and middle of bioreactor; maxdepth 12940 sequences per sample; 10 rarefactions. Bioreactor PositionGini Coefficient Observed OTUs Shannon Diversity Bottom 0.969 ± 0.004162 ± 14 4.603 ± 0.288 Middle 0.975 ± 0.006 159 ± 18 3.942 ± 0.560

TABLE 12 Gas Production Rate per period (for the main periods in theExample only an average rate across all periods is reported; P1 is notincluded because data was not available) Period Gas Production Rate (mLd⁻¹) Main (P2 to P7) 0.38 ± 0.01 P8 1.22 ± 0.06 P9 1.19 ± 0.03 P10 1.66± 0.27 P11 2.74 ± 0.04

Although the present disclosure has been described with respect to oneor more particular embodiments and/or examples, it will be understoodthat other embodiments and/or examples of the present disclosure may bemade without departing from the scope of the present disclosure.

The invention claimed is:
 1. A method for producing a productcomposition comprising 50% or greater by weight n-caprylic acid and/orn-caprylate comprising: a) providing a reaction medium comprising amicrobiome comprising Acinetobactor spp. having a pH of 5-7.5; b) addinga substrate comprising ethanol or a mixture of ethanol and acetate; c)holding the reaction medium at a temperature of 15° C. to 45° C. duringan acclimation phase until the reaction mixture produces a desiredamount of n-caprylic acid or n-caprylate; d) continuously removing atleast a portion or all of the n-caprylic acid or n-caprylate formed inthe reaction medium during the acclimation phase, wherein the reactionmedium is maintained at a pH of 5-7.5 during c) and, optionally d); ande) continuously removing during a production phase at least a portion orall of the n-caprylic acid or n-caprylate formed in the reaction mediumto form the product composition.
 2. The method of claim 1, whereinadditional substrate is added during the acclimation phase and/or duringthe production phase.
 3. The method of claim 1, wherein the substrate isethanol.
 4. The method of claim 1, wherein the substrate is a mixture ofethanol and acetate.
 5. The method of claim 4, wherein the ethanol andacetate molar ratio is 4.5 or greater.
 6. The method of claim 4, whereinthe ethanol and acetate molar ratio is 10 or greater.
 7. The method ofclaim 1, further comprising a selection period that is carried out aspart of the acclimation phase, subsequent to the acclimation phase, oras part of the production phase, wherein during the selection period non-caprylic acid or n-caprylate is removed from the reaction mixture. 8.The method of claim 1, further comprising a selection period that iscarried out as part of the acclimation phase, subsequent to theacclimation phase, or as part of the production phase, wherein duringthe selection period the concentration of n-caprylic acid and/orn-caprylate in the reaction mixture is greater than 0.1 g/COD/L.
 9. Themethod of claim 1, further comprising a selection period that is carriedout as part of the production phase, subsequent to the acclimationphase, or as part of the production phase, wherein during the selectionperiod no n-caprylic acid or n-caprylate is removed from the reactionmixture.
 10. The method of claim 1, further comprising a selectionperiod that is carried out as part of the production phase, subsequentto the acclimation phase, or as part of the production phase, whereinduring the selection period the concentration of n-caprylic acid and/orn-caprylate in the reaction mixture is greater than 0.1 g COD/L.
 11. Themethod of claim 1, wherein the reaction mixture is under ambientpressure.
 12. The method of claim 1, wherein the reaction mixture ispresent in an anaerobic environment.
 13. The method of claim 1, whereinafter the acclimation phase the reaction mixture produces n-caprylatecorresponding to at least 1 g chemical oxygen demand (COD)/L-d.
 14. Themethod of claim 1, wherein during the production phase the reactionmixture has a product ratio of n-caprylate to n-caproate of at least 10g COD/g COD.
 15. The method of claim 1, wherein the product compositioncomprises at least 50% n-caprylic acid and/or n-caprylate by weightbased on the total amount of product compounds in the productcomposition.
 16. The method of claim 1, wherein the microbiome does notcomprise Clostridium kluyveri during phase II of production ofn-caprylic acid or n-caprylate.
 17. The method of claim 1, wherein themicrobiome comprises Desulfosporosinus meridiei, Oscillospira spp,Burkholderia spp, and unknown Ruminococcaceae.